The Story of Stem Cells

 

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Chapter 3: Applications of Stem Cells in Medicine

Stem cell therapies carry immense potential for treating a number of human diseases. However, the demand for donor organs outweighs the availability by huge proportions. Another commonly faced challenge of organ transplants is the high incidence of rejection due to the recipient’s immune system. The application of stem cells for generating tissues and organ grafts holds tremendous promise to fulfill the largely unmet need for organ transplants and to improve the quality of life for millions of patients. Stem cells can be directed to generate specific cell types and can be used to replace ailing or degenerating tissues. The greatest advantage of this technique is that it overcomes graft-to-host incompatibility. Such tissues are generated from the patient’s own stem cells. Therefore, the chances of graft rejection by the host’s immune system decrease significantly. Stem cells have been employed in the treatment of spinal cord injuries, stroke, burns, diabetes, cardiovascular diseases (CVD), osteoarthritis, rheumatoid arthritis, and so on (Fig. 3.1) [1].

 

TICEBA - The Story of Stem Cells - Chapter 3 - Fig. 3.1 - Potential applications of stem cells in human disease

Fig. 3.1: Potential applications of stem cells in human disease [1].

 

TICEBA - The Story of Stem Cells - Chapter 3 - Fig. 3.2 - Steps involved in the development of stem cell therapy

Fig. 3.2: Steps involved in the development of stem cell therapy [2].

 

This diverse application of stem cells in therapeutics has triggered a surge in clinical trials investigating and validating stem cell based treatment options. The use of stem cells in therapeutics requires a series of systematically planned and executed steps that prove their therapeutic ability. Once a population of stem cells has been retrieved from the patient, it is directed to differentiate into a specific cell type with the help of certain factors or inducer cells [1]. A pure culture of the cell type of choice is then generated before its physiological functions are tested in vitro. The identity of these cells is then confirmed with markers that are specific to the cell type of interest. Attaining pure cultures is critical in stem cell therapy as undifferentiated stem cells have the ability to induce tumor formation when injected into the recipient. These tumors are known to benign in nature. Differentiated stem cells, however, do not pose such a risk of tumor formation [1]. The differentiated cells are then tested in a variety of animal models to prove their therapeutic potential before researchers proceed to human trials. Figure 3.2 depicts a schematic representation of the core steps involved in the development of stem cell therapy [2].

 

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3.1 Stem Cells in the Treatment of Cardiovascular Diseases

Cardiovascular diseases (CVD) include hypertension, coronary artery disease, stroke, and congestive heart failure. CVD are ranked as one of the major causes of death worldwide and are a significant health issue of the 21st century. CVD often result in the deprivation of oxygen supply to heart cells known as cardiomyocytes, leading to cardiomyocyte damage and narrowing of the blood vessels that supply the heart. Many survivors of heart attacks suffer from breathlessness and fatigue due to the extensive damage incurred by the heart muscles. Stem cell therapy could be a boon to these patients as it can help the heart heal itself. The fully developed human myocardium does retain some regenerative capacity. There is evidence that the adult myocardium contains a small population of cardiac stem cells with the ability to differentiate into cardiomyocytes and some other cell types [3,4]. This feature, however, is inadequate to compensate for the damage inflicted by myocardial infarctions or other catastrophic events that result in cardiac damage [5]. Restoration of damaged cardiomyocytes and vascular endothelial cells (cells that form the lining of the blood vessels) by stem cell therapy appears to be a promising treatment for this ailment. Skeletal muscle myoblasts, bone marrow–derived mononuclear cells, umbilical cord blood cells, mesenchymal stem cells, and cardiac stem cells have been used for cardiac tissue regeneration [13]. Animal models have been instrumental in establishing the safety and efficacy of these techniques. Stem cell therapy for CVD involves deriving cardiomyocytes from stem cells and injecting these cells into the damaged portion of the heart. These cells then integrate into the heart and secrete certain proteins and paracrine factors that further aid the repair of the damaged area (Fig. 3.3)[6]. Stem cell therapy has shown beneficial effects in animal models; however, the injected cells have low survival rates post-transplantation [7,8]. These observations are reason to believe that the improvement in cardiac health observed post stem cell injection might be due to certain trophic factors which are bioactive compounds like cytokines and growth factors secreted by cells and have autocrine and paracrine activities. These factors act indirectly by creating a micro-environment that will instigate a stem cell to develop into a particular type of differentiated cell [9]. The trophic factors help in the repair of the damaged tissue indirectly [9,10]. Researchers have also attempted to study the benefits of these secretory proteins as therapeutic options for cardiac regeneration post–myocardial infarction [6].

 

TICEBA - The Story of Stem Cells - Chapter 3 - Fig. 3.3 - Stem cells in repairing damaged cardiac tissue

Fig. 3.3: Stem cells in repairing damaged cardiac tissue [6].

 

TICEBA - The Story of Stem Cells - Chapter 3 - Fig. 3.4 - Options and challenges in stem cell therapy for cardiac regeneration

Fig. 3.4: Options and challenges in stem cell therapy for cardiac regeneration [7].

 

Cell Phase Condition Cell delivery route Basis of trial design Result
Autologous BM-MSCs II/III AMI Intracoronary Repairing the damaged myocardium via paracrine signaling Autologous BM-MSCs are safe and provide modest improvement in LVEF
Auto-hMSCs and allo-hMSCs I/II CILVD Transendocardial Prevention remodeling of the ventricle and reduction of infarct size Alloimmune reactions of allogeneic MSCs injection are low and improved functions are observed
Autologous BM-MSCs I/II Heart attack Intramyocardial Repair and restore heart function by reducing fibrosis, neoangiogenesis, and neomogenesis Autologous BM-MSCs could reduce scar, enhance regional function, and improve tissue perfusion
Allogeneic BM-MSCS I MI Intravenous Transdifferentiation of MSCs into cardiomyocytes Intravenous allogeneic hMSCs are safe in patients after AMI
Allogeneic BM-MSCS I/II MI Intravenous Transdifferentiation of MSCs into cardiomyocytes and production of new blood vessels Intravenous infusion of allogeneic BM-MSCs is safe and well-tolerated in AMI patients
WJ-MSCs II STEMI Intracoronary Transdifferentiation of MSCs into cardiomyocytes Intracoronary infusion of WJ-MSCs is safe and effective in patients with AMI
Autologous MSCs and BMCs I/II LVD Transendocardial Stimulation of endogenous cardiac stem cells by MSCs Transendocardial injection with MSCs or BMCs appeared to be safe for patients with ICM and LVD
AD-MSCs II CMI Not special Angiogenesis -
Autologous BM-MSC I/II CHF Intramyocardial Development of new myocardium and blood vessels Intramyocardial injections of Autologous culture expanded MSCs were safe and improved myocardial function

Table 3.1: Completed MSC-based randomized clinical trials for ischemic heart disease therapy registered at https://clinicaltrials.gov/ [11]

 

Figure 3.4 summarizes the different approaches and challenges involved in the application of stem cell therapy to repair an ailing heart [7]. Recent developments in clinical trials investigating the therapeutic applications of mesenchymal stem cells and the outcomes with different routes for the delivery of these cells in the treatment of CVD are detailed in Table 3.1 [11]. As part of the phase I clinical trial, CADUCEUS Study was conducted by the team at Cedars-Sinai Heart Institute in Los Angeles, USA. The results showed that an infusion of cardio sphere-derived stem cells from the patient’s own heart was used to help regrow damaged heart muscle cardiomyocytes.

The patients who underwent this treatment showed unprecedented improvement in viable heart mass and contractility in just six months. The study gained wide appreciation for its success. The CADUCEUS Study deemed autologous transfer of stem cells for the treatment of ventricular dysfunction a safe procedure warranting extension of the study into phase II [12]. The pharmacological and surgical approaches leave behind scars of damaged cardiac tissue. The promise of stem cell therapy for treating ischemic heart diseases is now a feasible therapeutic option. The POSEIDON, SCIPIO, SWISS-AMI, CADUCEUS, and CCTRN trials as well as the TIME, LateTIME, and FOCUS clinical trials have been instrumental in demonstrating the efficacy and safety of mesenchymal, cardiac, and bone marrow–derived mononuclear cells in reducing infarct size and improving heart muscle contractility. Preclinical studies have now indicated that a cocktail of cardiac and mesenchymal stem cells could be more effective in restoring cardiac function. Thus, studies exploring more efficient delivery systems, the optimal combination of stem cells, and the mechanism orchestrating the beneficial effects observed, are being pursued [13-19].

 

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3.2 Stem Cells in Bone Regeneration

This chapter will begin with a brief introduction about the key components of the human skeletal system. The skeletal system forms a support mechanism for the whole human body. It provides contour, strength and helps in all our movements whether it is moving around, lifting a book from the table, or even drinking a glass of water. We all depend on the joint function of muscles and bones for our day to day activities. Thus, ailments of the bone have drawn attention from medical professionals and scientists, who work hard to improve the quality of human life. This chapter will address the components of the skeletal system, followed by factors, and the recent developments in the field of stem cell treatments.

The human skeletal system is made up of approximately 206 bones, as well as an extensive network of tendons, ligaments, and cartilage that connects them. Ligament is a fibrous band of tough connective tissue, which holds bones at joints. Tendon is also a fibrous connective tissue and connects muscles to bones. They are capable of withstanding tension. Cartilage is a connective tissue that forms a protective cover over the surface of joints helping a smooth movement. Since cartilage is devoid of blood supply and nerve connections, any incurring damage does not heal quickly. The lack of nerve supply also means that cartilage damage does not cause pain, at least not until the cartilage in that area is completely damaged and the bone gets affected. Cartilage damage can occur due to sudden injuries such as a fall, an accident or sport related injuries, osteoarthritis or infection.

Contrary to its hard and stable appearance, the bone is a metabolically active organ and exists in a constant state of dynamic turnover, known as bone remodeling. There are two main cell types in this process: osteoclasts and osteoblasts. Osteoclasts are bone resorbing cells, mainly involved in the removal of mineralized bone. They are the bone destroying cells. Osteoblasts are connective tissue cells found on the surface of bone that are actively involved in the formation of the bone matrix. Osteoblasts can be stimulated to proliferate and differentiate into bone cells known as osteocytes. Osteocytes are enclosed in bone and they manufacture type 1 collagenase and other substances that form the extracellular matrix of bone.

Any imbalance in the bone remodeling process can result in damage to the bone structure, thus is prone to the individual susceptible to diseases such as osteoporosis [20]. Congenital malformations, tumor resections, fractures, trauma as well as diseases such as arthritis can result in bone damage [20]. Old age is also one of the leading causes of bone related ailments. Autologous bone grafting has been the gold standard approach for treating large bone defects. This procedure includes harvesting a graft from healthy tissue and transplanting it into the damaged site. Though better than most available treatment options, the success of this approach is mainly restricted as it causes injury to the area from which the bone is extracted (donor site morbidity), inflammation, and resorption of the transplanted bone. The advent of stem cell technology has paved the way for novel strategies to tackle this challenge.

Mesenchymal stem cells/progenitor cells (MSCs) are found in adult bone marrow, adipose tissue, skin, umbilical cord, and the placenta. They have multi lineage differentiation capabilities. These capabilities are osteogenic, chondrogenic, and adipogenic. Osteogenic can give rise to bone cell osteoblasts. Chondrogenic can give rise to muscle cell chondrocytes and adipogenic can give rise to fat cells. They all play a key role in bone remodeling [21,22] (Fig 3.5).

MSCs can be used for a wide range of therapeutic applications, including bone and cartilage repair, as they play an important role in bone repair [23]. Quarto et al were the first to publish evidence towards the use of MSCs for filling large bone defects about 4-7cm in length. The group was able to successfully repair the tibia, humerus, and ulna of 3 individual patients, respectively [24].

The first step in bone tissue engineering is harvesting bone marrow from the patient. MSCs are extracted from the bone marrow by adhering them on plastic culture plates. The purified MSCs fraction is then allowed to proliferate. The proliferated cells are further seeded on to a synthetic scaffold to allow in vitro growth over several days or weeks. This allows for scaffold colonization and for cell differentiation before grafting the processed composite material at the affected site into the same patient [25,26] as shown in Fig 3.6. Studies have also explored the possibility of pharmacological stimulation of MSCs to direct them to differentiate into osteoblasts, which are the bone forming cells. For example, Bortezomib, a clinically available proteasome inhibitor, induced MSCs to successfully differentiate into osteoblasts and thus enhanced the bone formation and rescued bone loss in a mouse model of osteoporosis [27]. Bone marrow has been the classic source of MSCs, however, the pain and morbidity involved in bone marrow extraction makes it a less preferred option. Adipose tissue, on the other hand, is being considered as an ideal source for a high yield of MSCs due to its ubiquity, ease of retrieval, and the minimally invasive procedure that used for its extraction. Such stem cells are termed adipose tissue derived MSCs (ASCs).

 

TICEBA - The Story of Stem Cells - Chapter 3 - Fig. 3.5 - TGF-β1 induces migration of MSCs to the bone remodeling sites to couple bone resorption and formation

Fig. 3.5: TGF-β1 induces migration of MSCs to the bone remodeling sites to couple bone resorption and formation. The bone-resorptive microenvironment also provides signals that direct the cell lineage–specific differentiation of MSCs [21].

 

ASCs are capable of inducing bone regeneration and repair as evidenced by in vitro and in vivo studies [28]. Bone healing in this case is induced by either direct differentiation into osteoblasts or via paracrine effects that facilitate the migration and differentiation of resident progenitor cells. For example, vascular endothelial growth factor (VEGF), an adipokine secreted by ASCs, activates the formation of a new network of blood capillaries necessary for bone regeneration [29]. VEGF also acts directly by recruiting hematopoietic stem cells for bone regeneration [28]. A large amount of preclinical data derived from experimentation on animal models has ascertained the bone healing properties of ASCs, a brief account of which is compiled in Table 3.2 by Barba et al [28]

 

TICEBA - The Story of Stem Cells - Chapter 3 - Fig. 3.6 - Bone tissue engineering from stem cells

Fig. 3.6: Bone tissue engineering from stem cells.

 

Translating preclinical data into clinical practice entails manipulating human tissues for the purification and production of clinical-grade ASCs to be employed as therapeutic devices. These procedures are strictly governed by the guidelines laid down by regulatory bodies such as the US Food and Drug Administration and the European Union Medicines Agency, which guarantee the safety and quality of the products [30]. Table 3.3 summarizes the clinical trials that are currently being undertaken to establish the application of stem cells in bone regeneration [31].

 

Experimental model Species Scaffold/ Administration Additional ex vivo/in vivo treatment Graft type
Calvarial defect Rat Rabbit Mouse Rat Mouse Dog Mouse Mouse Rat Rat PLGA HA-PLGA, collagen sponge PLGA β-TCP Custom scaffold HA-PLGA Systemic injection Local injection DBM, PLA MAP-coated PCL/PLGA Alendronate BV-BMP2/TGF β3 Dura mater Lenti-miR-31 Noggin shRNA-Knockout None None None None None Xenogenic Allogeneic Xenogenic Allogeneic Xenogenic Xenogenic Allo/xenogenic Xenogenic Xenogenic Xenogenic
Calvarial defect Rat Rat Dog Dog Pig Rat Rat Mouse Rabbit Mouse Rat HA-β-TCP PLGA Coral Coral Collagen sponge DBX PCL-PLGA-β-TCP pDA-PLGA Collagen sponge HA-PLGA Local injection None None/osteogenic medium Osteogenic induction Osteogenic induction Osteogenic induction Osteogenic induction Osteogenic induction+HUVEC rhBMP-2 rhBMP-2 Sonic hedgehog signaling Induction VEGFa Xenogenic Xenogenic Autologous Allogeneic Autologous Allogeneic Xenogenic Xenogenic Allogeneic Xenogenic Xenogenic
Ectopic bone formation Mouse Mouse Mouse Rat Rat Rat Mouse Rat PLGA PRP+alginate microsphere Β-TCP HA Matrigel DBM Carbon nanotubes PLDA BMP2/RUNX2 bicistronic vector None None None Osteogenic induction Osteogenic induction rhBMP2 rhBMP2 Xenogenic Allogeneic Xenogenic Xenogenic Xenogenic Xenogenic Xenogenic Xenogenic
Segmental defect Rabbit Rat Rat Rabbit Rabbits Local injection Fibrin matrix β-TCP PLA/PCL+vascularized periosteum HA-PLA-COL Bovine BMP rhBMP2 Lenti-BMP2/7 Ad-Cbfal Ad-hBMP2 Allogeneic Allogeneic Allogeneic Allogeneic Allogeneic
Segmental defect Mouse Rat Rabbit Dog Rabbit Rabbit Systemic injection Collagen gel PLGA β-TCP HA Ceramics, biphasic materials None None None/osteogenic medium None None None Allogeneic Xenogenic Xenogenic Allogeneic Autologous Allogeneic
Vertebral defect/fusion Mouse Rat Rat Local injection Lyophilized human cancellous bone Fibrin gel rhBMP6 nucleofection Gal-KO+osteogenic induction rhBMP6 nucleofection Xenogenic Xenogenic Xenogenic
Mandible defect Pig Rat Local-systemic injection HA/COL None None Allogeneic Xenogenic

Table 3.2: Preclinical studies on ASC osteoregenerative potential. Source: Barba et al [28]

 

Study title Conditions Intervention Cell preparation Estimated enrollment Study design
Percutaneous Autologous bone marrow grafting for open tibial shaft fracture (IMOCA) (NCT00512434) Open tibial fractures Standard of care, with percutaneous injection, 1 month after fracture, of Autologous concentrated bone marrow to defect site Concentrated BMA 85 Randomized, parallel assignment, open label
Mesenchymal stem cell for osteonecrosis of the femoral head (NCT00813267) Osteo-necrosis of femoral head Infusion of BMSC into the femoral artery Ex vivo culture 15 Single group
Distraction osteogenesis in limb length discrepancy with mesenchymal cell transplantation (NCT01210950) Leg length inequality Injection of BMSC with plasma-rich protein into callus Not specified 6 Single group
Mesenchymal stem cells; donor and role in management and reconstruction of nonunion fracture (NCT01626625) Nonunion fracture Transplantation of Autologous BMSC seeded upon a hydroxyapatie scaffold Ex vivo culture 10 Parallel assignment, double blind
Clinical trial based on the use of mesenchymal stem cells from autologous bone marrow in patients with lumbar intervertebral degenerative disc disease (NCT01513694) Intervertebral disc disease Instrumented posterolateral fusion with Autologous BMSC on a phosphate ceramic Ex vivo culture 15 Single group
Treatment of maxillary bone cysts with Autologous bone mesenchymal stem cells (NCT01389661) Maxillary cyst Transplantation of Autologous BMSC seeded upon an Autologous plasma protein matrix into cyst cavity Seeding on scaffold, ex vivo culture 10 Single group
Safety study of mesenchymal stem cells and spinal fusion (NCT01552707) Lumbar spondylolisthesis involving L4-L5 Instrumented spinal fusion combined with autologous BMSC on allogeneic bone graft Ex vivo culture 62 Randomized, parallel assignment, open label
Mesenchymal stem cells in osteonecrosis of femoral head (NCT01605383) Avascular necrosis of femur head Core decompression combined with implantation of Autologous BMSC on allogeneic bone graft in lesion Ex vivo culture 24 Randomized, parallel assignment, open label
Treatment of osteonecrosis of the femoral head by the administration of autologous mesenchymal stem cells (NCT01700920) Osteonecrosis of the femoral head Intraosseous injection of Autologous BMSC with trocar in the femoral head Ex vivo culture 10 Single group
The efficacy of mesenchymal stem cells for stimulate the union in treatment of non-united tibial and femoral fractures in Shahid Kamyab Hospital (NCT01788059) Nonunion Fracture Percutaneous injection of Autologous bone marrow mononuclear cells into defect BMA mononuclear fraction 18 Single group
Evaluation of the efficacy and safety of Autologous MSCs combined to biomaterials to enhance bone healing (NCT01842477) Delayed union after fracture of humerus, tibial or femur Implantation surgery of a synthetic bone substitute associated with Autologous BMSC Ex vivo culture, osteogenic differentiation, seeding on TCP scaffolds 30 Single group
Treatment of atrophic nonunion fractures by Autologous mesenchymal stem cell percutaneous grafting (NCT01429012) Nonunion fracture Percutaneous injection of BM into the nonunion space Not specified 40 Parallel assignment, double blind
Mononucleotide autologous stem cells and demineralized bone matrix in the treatment of nonunion/ delayed fractures (NCT01435434) Nonunion fracture Transplantation of Autologous bone marrow mononuclear cells with demineralized bone matrix BMA mononuclear fraction Not stated Single group assignment

Table 3.3: Ongoing clinical trials employing skeletal stem cell containing populations for bone regeneration. Source: Dawson et al [31]

 

Recently, the research team headed by Dr. Barbara Chan at the University of Hong Kong was able to generate new cartilage tissue in the lab from a patient’s own stem cells. The team pioneers in cartilage tissue engineering. This technology can be used in the treatment of sport related injuries and those caused by sudden trauma. The team is working to extrapolate these findings to the treatment of other cartilage diseases such as osteoarthritis and degeneration [32,33].

Thailand is also another country on its way to become a leader in stem cell research. The Police General Hospital, Bangkok, Thailand has been conducting a clinical trial with 60 arthritis patients using stem cells. The team headed by Pol.Mai.Gen Dr. Thana Turajane derived cartilage from stem cells extracted from blood which are being used in research [34]. In 2012, the research team at Mahidol University's Siriraj Hospital also reported the successful extraction of pure stem cell population from amniotic fluid and predicted its potential use to treat several severe conditions including arthritis [35].

Recently, researchers at the Stanford University School of Medicine discovered a new type of mouse skeletal stem cell that can differentiate into bone, cartilage, and stromal cells [36]. The group was also able to define the chemical signals that can be potent inducers of osteogenesis. If these findings are translatable to humans it would immensely benefit patients with osteoporosis and osteoarthritis. The odds appear to be in favor of this being a successful therapeutic option in human subjects, as the mouse and human skeletal systems share many similarities in terms of genetic makeup. Cartilage regeneration has been one of the main challenges in tissue regeneration thus far. Any tear in cartilage has to be treated surgically by removing the torn tissue, which predisposes the patient to arthritis at a later period. Based on these findings, it is now possible to derive new cartilage from the patients’ own stem cells and treat this condition effectively [37].

Osteogenesis imperfecta (OI) is an incurable congenital disease, commonly known as brittle bone disease. It is a genetic condition caused by mutation of the gene that code for the protein collagenase. OI causes multiple bone fractures in fetuses in utero (inside the womb) and these infants die shortly after birth. Those that survive are faced with a life of brittle bones, falling teeth, and stunted development. A multicentric study, initiated in January 2016, is attempting the use of stem cells to cure OI and is being coordinated by lead researchers at Sweden’s Karolinska Institute in collaboration with leading research centers in Europe including the University of Leicester. "It is the first in-man trial and, if successful, it will pave the way for other prenatal treatments when parents have no other option" said Dr. Gotherstrom from the Karolinska Institute [38].

European Union Horizon 2020 is a framework programme of the European Union for research and innovation that recently funded the NUI Galway Regenerative Medicine Institute’s (REMEDI) of which the most recent endeavor is called AUTOSTEM. AUTOSTEM is an interdisciplinary research venture to develop a state of the art facility for robotic stem cell production. This project will offer innovative stem cell based therapeutic options for a range of diseases. REMEDI is a European leader in therapeutic stem cell research and houses scientists that lead many EU funded programs focused at developing stem cell based treatments for diseases like osteoarthritis, diabetes, and corneal transplantation [39]. Stem cell treatments still hold the promise for a better life to bone disease patients.

 

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3.3 Stem Cells in the Treatment of Eyesight

Vision is most likely the most important sense with which we perceive the world. Lack of eyesight can have a dramatic effect on a person and plunge his or her world into darkness. As most diseases affecting the eye are difficult to treat, stem cell technology is currently being explored to restore the gift of sight to patients, who lost their vision due to various reasons. This section will begin with a brief description of the important parts of the eye, which will help in understanding the terminologies used in this article. We will then learn about the most common diseases affecting vision and how developments in stem cell therapy help patients to regain their vision. This segment will also brief on the current stem cell based treatment options available for treating these conditions.

In order to obtain a better picture of these developments, it is first necessary to gain some basic knowledge of the structure and functional characteristics of the eye. The eye is a complex organization of specialized components that comprise the cornea, iris, retina, lens, macula, and a dense blood and nerve supply network to aid in its functioning.

 

TICEBA - The Story of Stem Cells - Chapter 3 - Fig. 3.7 - The human eye

Fig. 3.7: The human eye. Inset 1 illustrated various layers of the retina. The outer nuclear layer (ONL) and the outer segments (OS) are located adjacent to the monolayer of Retinal pigment epithelium (RPE) cells. Source: Jha and Bharathi [40].

 

The cornea is a transparent protective covering of the iris, which we commonly refer to as eye color. The transparency of the cornea is maintained by constant renewal of the corneal epithelial cells by stem cells in the limbus, the region of the cornea bordering the sclera. The iris controls the dilation of the pupil and thus regulates the amount of light reaching the retina.

The retina is the light sensitive part of the eye. It consists of a thin layer of tissue that covers about 65% of the eye and its main function is to convert light into neural signals and communicate them to the brain via the optic nerve. The retina contains photoreceptor (PR) cells called rods and cones that are responsible for sensing color and light intensity. The retinal pigment epithelium (RPE) is an essential supporting tissue and provides nutrition to the light sensitive cells of the retina. It is also responsible for recycling retinol, the production of pigment and phagocytosis of photoreceptor cells (PR) outer segments (OSs) of the rods and cones [41]. The RPE tissue is composed of a monolayer of polarized RPE cells that are situated on a proteinaceous Bruch’s membrane (Inset I in Fig. 3.7). The RPE is the thin black line that we see surrounding the retina. Any damage to the RPE cells renders PR cells of the retina vulnerable to damage and can result in their rapid degeneration.

The macula is the most vital part of the retina; it is located at the back of the eye and is vital for sharp, central vision. The macula plays an important role in activities such as driving, reading, and face recognition. Loss of vision can be caused by damage of any part of the eye such as the retina, cornea, or the optic nerve. A few of the most common causes of blindness and the current stem cell therapy options are discussed below.

Age-related macular degeneration (AMD) is the most common cause of impaired vision due to retinal degeneration in old age and affects over 40 million people worldwide [42]. As the name suggests, AMD is caused by a gradual degeneration of the macula due to the aging process. Blurring of the central vision is a common symptom associated with AMD (Fig. 3.8 B). There are two types of AMD: dry form and wet form. In the dry form, yellow deposits called drusen are found in the macula that increase in size and number over time, causing blind spots and distorted vision. The wet form of macular degeneration is caused by the abnormal growth of blood vessels in the macula, which leak blood and fluid into the retina, thereby hampering vision. These blood vessels and their bleeding eventually cause the loss of central vision (Fig. 3.8B). The dry form of AMD is more commonly seen when compared to the wet form [43].

Stargardt macular dystrophy (SMD) is an autosomal recessive, genetically inherited form of macular degeneration caused by a mutation in the ABCA4 gene. ABCA4 codes for a protein that aids in the removal of potentially toxic substances, such as lipofuscin that are formed in PR and OS cells when PR cells convert light into electrical signals for the brain to interpret. Under normal conditions, the RPE brings about the phagocytosis of millions of PR and outer OS cells. The incomplete digestion of phagosomes (a vesicle formed around a particle absorbed by phagocytosis) results in the accumulation of lipofuscin, which causes the degeneration of RPE cells, in turn causing the death of PR cells and the progressive loss of vision. The symptoms of SMD usually appear in late childhood or early adulthood as central vision fails over time (Fig. 3.8B). Although the etiology of this disease is well established, there is no recognized cure for SMD [41,44].

 

TICEBA - The Story of Stem Cells - Chapter 3 - Fig. 3.8 - Effects of damage to RPE cells in visual field

Fig. 3.8: Effects of damage to RPE cells in visual field. (A) Normal Vision (B) Macular dysfunction as in the case of Age related macular degeneration (AMD) and Stargardt macular dystrophy (SMD) (C) The tunnel visual field experienced in individuals with retinitis pigmentosa.

 

Retinitis pigmentosa (RP) is also an inherited degenerative retinal disease with no known cure. RP is mainly caused by abnormalities in PR cells or the RPE resulting in progressive loss of vision. Patients initially experience night blindness followed by the loss of peripheral vision (commonly known as tunnel vision) (Fig. 3.8C), which is often followed by blindness [45]. Deposition of a pigment on the peripheral regions of the retina is a classic signature of RP.

Optic nerve hyperplasia (ONH) is a congenital abnormality in the development of the optic nerve and is one of the major causes of blindness among children. The optic nerve transmits information from the retina to the brain. Children with ONH also tend to have brain and pituitary malformations. ONH can affect either one eye or both eyes and is not a progressive disease. Thought to be incurable, patients with ONH now hope to see again due to the advent of stem cells [46].

Damage to RPE cells appears to be the main pathology behind AMD, SMD, and RP. The RPE cells and the retina lack intrinsic regenerative capabilities and therefore cell replacement was always considered a potential therapeutic option [2]. Autologous transplantation to replace RPE was thought to be beneficial to these patients. However clinical trials that attempted this approach were unable to show functional improvements mainly due to immune rejection and graft failure [47]. The regenerative capacity of stem cells makes them an ideal source for the derivation of specialized cells that could be used to repair damaged portions of the eye causing impairment or complete loss of vision. The retina in particular is often considered an excellent target for a stem cell therapy, as it can be easily monitored for improvements of the post treatments [41].

Mesenchymal stem cells (MSC), neural stem cells (NSC), human embryonic stem cells (hESC), induced pluripotent stem cells (iPSC), endogenous retinal stem cells such as Müller glia, RPE stem cells, limbal stem cells (LSC), and ciliary epithelium-derived stem cells have been used in the treatment of retinal degeneration [48]. RPE cells derived from hESC have been shown to rescue PR cells in animal models [49,50]. However, their use is restricted due to obvious ethical reservations.

Studies involving bone marrow-derived hematopoietic stem cells (BMHSC) delivered via injection into the tail vein of mice with experimentally induced retinal damage have shown evidence that these cells migrate to the retina and express RPE65, a RPE-specific protein [51]. A California-based research group is also attempting intravitreal (inside the eye) administration of BMHSC to treat retinal occlusion after the procedure was ascertained to be safe in animal models [52]. Improvement in RPE and PR morphology has also been reported in mouse models of RP after sub-retinal injections of bone marrow-derived mesenchymal stem cells (BMMSC) [53].

PR cells and RPE cells have been successfully derived from human induced pluripotent stem cells (hiPSC) [54]. These hiPSC-derived RPE cells were able to form monolayers of RPE cells, effectively remove toxic substances such as lipofuscin by phagocytosis, express genes vital to the visual cycle, and improve visual function when transplanted into the Royal College of Surgeons (RCS) rats [55,56]. The RCS rat is a commonly used animal model for RPE diseases, including AMD. The world’s very first clinical trial using iPSC cells for the treatment of AMD patients was initiated by a Japanese group at the RIKEN research center in 2014 [57,58]. This study was however suspended after the iPSC generated from the second patient did not pass the genomic validation tests and contained a possible oncogenic mutation [59]. Therefore, the use of iPSC still remains unclear and their safety needs to be thoroughly investigated in preclinical models before being considered for human therapeutics.

Preliminary observations from a clinical trial conducted by Schwartz et al in 2012 reported significant improvement in vision after transplanting hESC-derived RPE cells into two patients (one with AMD and the other with SMD). This group was the first to demonstrate the clinical safety of hESC-derived RPE cells for transplantation into human subjects [60]. Song et al have also reported the safety and tolerability of hESC-derived RPE transplantation in a study conducted on four Asian patients. No evidence of adverse proliferation, tumorigenicity, ectopic tissue formation, or any other abnormalities were observed. The patients experienced improvement in vision and were stable over a one- year follow-up period [61]. Stem cell therapy for curing retinal diseases is now moving into phase I/II clinical trials. Details on some of the ongoing clinical trials on this front are briefly listed in the Table 3.4.

Limbal stem cell deficiency (LSCD) is a result of the depletion or destruction of limbal stem cells (LSC) and can cause blindness in severe cases. Genetic conditions such as aniridia or dyskeratosis congenita, chemical or thermal burns, eye surgery or contact lens wear are listed as causes of LSCD [62]. Autologous LSC were shown to have successfully and permanently restored a transparent, self-renewing corneal epithelium in 76.6% of eyes in a study that involved 112 patients with corneal damage due to burns. The restored eyes were stable over a 10 year follow-up period without any adverse outcome [63]. A phase II clinical trial will soon be initiated in China to evaluate the potential of bone marrow-derived mesenchymal stem cells for treating patients with corneal burns [64].

China appears to be the hotspot for medical tourism as an increasing number of parents of children with ONH are pursuing stem cell therapy there. Beike Biotech is one of the leading companies in China offering donor cord blood-derived mesenchymal stem cells as a treatment for patients with ONH. The patients receive stem cell infusions into the cerebrospinal fluid over a period of two weeks. Several patient blogs vouch for the improvement in vision following this treatment [65,66]. The main concern that ophthalmologists and scientists have regarding this treatment is the lack of scientific literature that supports this claim. It is cautioned that patients resort to this treatment at their own risk and thorough investigation in terms of preclinical as well as well-structured clinical trials need to be conducted prior to implementing this treatment [46].

 

Disease Institution Cell source Delivery Immuno-suppression BCVA WHO identifier
STGD and AMD (GA) Advanced Cell Technology, USA hESC-derived RPE Subretinal injection of suspension postvitrectomy Yes: tacrolimus 20/40 0 NCT01469832
AMD University College London and Pfizer, UK hESC-derived RPE monolayer Subretinal on a plastic polymer patch postvitrectomy Intraocular steroid N/A NCT01691261
AMD (GA) Stem Cells Inc., USA Neuralised human foetal stem cells Subretinal Injection post-vitrectomy Yes: unspecified 20/40 0 NCT01632527
AMD (GA) Janssen R&D, USA hUTSC Micro-catheter via sclera and choroid N/A 20/20 0 NCT01226628
AMD (GA), RP and ischaemic retinopathy University of Sao Paolo, Brazil Autologous BMHSC Intravitreal injection N/A 20/20 0 NCT01518127 NCT01560715 NCT01518842
AMD (GA), RP, RVO and DR University of California, Davis, USA Autologous BMHSC Intravitreal injection N/A 20/20 0 NCT01736059
RP Mahidol University, Thailand BMMSC Intravitreal injection N/A 6/120 NCT01531348
DR Tehran University of Medical Sciences, Iran Autologous BMMSC Intravitreal injection N/A N/A IRCT201111 291414N29
DR General Hospital of the Chinese People’s Armed Police Force, China BMMSC N/A N/A N/A ChiCTR-TNRC -11001491
AMD and RP All India Institute of Medical Sciences, India Autologous BMHSC Intravitreal injection N/A 10/20 0 CTRI/2010/ 091/000639
AMD: age-related macular degeneration; BCVA: best corrected visual acuity; BMHSC: bone marrow-derived haematopoeitic stem cell; BMMSC: bone marrow-derived mesenchymal stem cell; DR: diabetic retinopathy; GA: geographic atrophy; hESC: human embryonic stem cell; hUTSC: human umbilical tissue-derived stem cells; N/A: information not available; RP: retinitis pigmentosa; RPE: retinal pigment epithelium; RVO: retinal vein occlusion; STGD: Stargardt’s disease.

Table 3.4: Clinical trials using stem cells for retinal disease that are currently registered on WHO clinical trial register. Source: Ramsden et al 2013

 

RP affects one in 4,000 people in the US and Europe and one in 1,000 people in China. About 100,000 people in the Philippines alone are affected by this condition [67]. The Institute of Personalized Medicine, Asian Stem Cell Institute, Makati Medical Center-Cellular Therapeutics Center (CTC), and Lung Center of the Philippines-Molecular Diagnostics and Cellular Therapeutics Laboratory recently received accreditation from the Department of Health (DOH) to provide stem cell-based therapies, including corneal resurfacing with LSC in the Philippines. The CTC is a Clean Room ISO class V facility and is also approved by the US Food and Drug Administration (US FDA). It also houses CliniMACS, a German made USA FDA approved cell sorting device that allows the facility to extract stem cells with over 90% purity based on specific cell markers [68,69].

 

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3.4 Stem Cells in the Treatment of Diabetes

Diabetes mellitus is one of the most prevalent diseases of the 21st century. Etiology of diabetes has been accredited to genetic predisposition, sedentary lifestyle, or environmentally incurred islet cell damage. Diabetes is also one of the most extensively studied diseases in the world. With an unprecedented rise in individuals suffering from diabetes and currently incurable, all present treatment options are focused on merely managing this condition. However, this scenario has not deterred scientists from looking for a cure. With their extensive applications and self-renewal properties, stem cells have been considered as the answer to finding a cure for diabetes. Before diving into the abundant literature available on this topic, this chapter will first address a brief description of the pancreas, basic causes, and the mechanism behind diabetes and discuss further detail of the development of stem cell therapy. We can see how much the world has achieved in combat against this disease.

The pancreas is a complex organ and consists of two main functional components, namely the exocrine cells and the duct cells that have both exocrine and endocrine functions. The exocrine cells constitute 90-95% of the organ, while the endocrine cells constitute 1-2%. Five distinct types of endocrine cells are present in the pancreas and each of them is specialized to produce specific pancreatic hormones, which are responsible for glucose homeostasis in the body. They are the alpha-cells (α-cells), beta-cells (β-cells), delta-cells (△-cells), PP-cells, and ε-cells. α-cells secrete glucagon and β-cells produce insulin. △- cells produce somatostatin, PP-cells secrete pancreatic polypeptide and ε-cells produce ghrelin.

Diabetes represents a significant consequence of a group of conditions commonly referred to as "the Metabolic Syndrome". Metabolic syndrome is a disorder that typically involves a cluster of conditions such as high blood pressure, obesity, and high blood sugar, which are linked to an increased risk of heart disease and type 2 diabetes. Diabetes is characterized by abnormally high glucose levels in the blood stream and inadequate levels of insulin. Clinical diagnosis of diabetes is established when a patient exhibits consistent hyperglycemia or high blood glucose, where the blood glucose levels raise above 126 mg/dL after an overnight fast [70]. In a healthy individual, insulin is secreted by β-cells present in the islet of Langerhans in the pancreas. β-cells are located closely to blood vessels, and thus respond to the change of blood glucose levels by secreting appropriate amount of insulin as shown in Figure 3.9A [71]. Tissue can lose its ability to regulate glucose due to a disturbance in either the amount of insulin or a malfunctioning of insulin receptors on cell surfaces. Thus, the cellular uptake of glucose in hampered and so the glucose levels in the blood remain high at all times.

Depending on its etiology (the causative factor), diabetes is classified into type 1 and type 2 diabetes. In type 1 diabetes, the β-cells of the pancreas are destroyed by the patient’s immune system, thereby resulting in absolute insulin deficiency as shown in Figure 3.9B. Type 1 diabetes is commonly known as juvenile onset diabetes as it affects individuals at a very early age. Type 1 diabetes is also known as insulin dependent diabetes as the patients have to constantly monitor their blood glucose and accordingly take regular injections of insulin several times a day. Type 2 diabetes mainly affects individuals at later stages of life mainly due to a sedentary lifestyle, obesity, and a genetic predisposition due to family history. In type 2 diabetes, the body becomes insensitive to insulin. In the initial stage, the pancreas produces enough insulin but with constantly high glucose levels due to malfunctioning of insulin receptors, the production over time is reduced. By the time diabetes is diagnosed, more than 80% of pancreatic β-cells would have failed in their function to produce Insulin. Failure of β-cells results in insulin deficiency which is termed Diabetes [70]. Thus, type 2 Diabetes is commonly referred to as insulin resistance. Due to its chronic nature, diabetes has widespread consequences on an individual’s health. Persistent hyperglycemia causes an array of complications that result in impairment in vision (Diabetic retinopathy and cataract), cardiovascular diseases, nerve damage (Diabetic neuropathy), renal failure (Diabetic nephropathy), and diabetic foot ulcers. Macro and microvascular diabetic complications also impede wound healing, which often results in gangrene and amputations [70]. A strict diet control, constant monitoring of one’s blood sugar levels, and a stringent exercise regimen to keep calories in check alongside with daily medications are just a few things that encompass the routine of a diabetic person, who manages diabetes for life and tries to stay healthy.

According to the National Institute of Health (NIH), approximately 1,300 people with type 1 diabetes receive whole pancreatic transplants in the USA and 83% of these patients do not show any symptoms of diabetes and also do not need insulin administration. However, organ transplant demands outweigh their availability. This approach also renders the patients predisposed to other disease as they are prescribed heavy doses of drugs that suppress the immune system to avoid organ rejection [72]. Alternatively, doctors have attempted injections of islet cells directly to the pancreas of type 1 diabetes patients to restore insulin production. This approach also required immunosuppressants to avoid auto destruction of the transplanted cells by the patient’s immune system [73].

 

TICEBA - The Story of Stem Cells - Chapter 3 - Fig. 3.9A - Insulin secretion by β-cells in the pancreas in a healthy individual

Fig. 3.9A: Insulin secretion by β-cells in the pancreas in a healthy individual [71].

 

TICEBA - The Story of Stem Cells - Chapter 3 - Fig. 3.9B - Type 1 and type 2 diabetes

Fig. 3.9B: Type 1 and type 2 diabetes. Type 1 Diabetes occurs as a result of β-cell destruction by immune cells, such as macrophages and T cells, whereas type 2 diabetes occurs as a result of damaged insulin receptors, thereby causing insulin resistance in the peripheral tissues (pancreas, muscle, fat, and liver cells).

 

With advances in stem cell technology there is a great interest in developing β-cells from stem cells, thereby helping the damaged pancreatic environment to repopulate itself and restore full functionality. The most evident place to look for these cells would be the pancreas. The regenerative ability of the pancreas has been well demonstrated in rodent models, where significant regeneration has been observed subsequent to 90% pancreatectomy (surgical removal of the pancreas) [74]. However, the fact that the pancreas houses its own stem cells is still a matter of debate [75]. Alternatively, other stem cell based approaches have been sought after and are listed in Table 3.5, and discussed at length below.

 

Source of cells Advantages Disadvantages Autologous Insulin pro- duced in vitro Insulin production in vivo (mice)
Pancreatic stem cells - Theoretical: already partially differentiated toward β-cells - Difficulty isolating cells and trans-differentiation factors Yes Yes No
Bone marrow stem cells - Established harvesting protocols - Immunoprotective qualities (suppress β-cells specific T-cells) - In vivo increases in insulin even without differentiation into β-cells - Stimulate β-cell regeneration in damaged pancreatic tissue - Difficulty replicating early differentiation results Yes Yes Yes
Embryonic stem cells - Potentially unlimited supply - Risk of teratoma development - Ethical controversy - No long-term studies - Difficulty with in-vitro work No Yes Yes
Induced pluripotent stem cells - Potentially unlimited supply, while avoiding ethical issues of embryonic stem cells - Retrovirus use and host genome integration (overcome with new derivation methods) Yes Yes Yes
Spermatogonial stem cells - Potentially unlimited supply - Avoids ethical issues of embryonic stem cells - No exogenous genes needed to induce pluripotency or differentiation into β-cells - No long-term studies - Short-term male centric - Long-term information may be applied to female ovarian stem cells Yes Yes Yes

Table 3.5: Summary of selected stem cell-based therapies for type 1 diabetes [76].

 

Soria et al were the first to derive insulin producing cells from embryonic stem cells (ESC) in mice. When these insulin producing cells were transplanted back into experimentally induced diabetic mice, they were able to reverse the hyperglycemic condition in these animals [77]. Despite the resemblance between ESCs derived insulin secreting cells and β-cells, these cells failed to exhibit insulin encapsulated secretary granules, which is a characteristic feature of β-cells [78]. Moreover, ESCs are known to cause teratomas in humans [79]. The risk of teratomas post transplantation of stem cells can be overcome by increasing their differentiated state and sorting differentiated cells from the general cell population using flow cytometry prior to the transplantation [80]. A flow cytometer separates cells based on specific marker proteins present on the surface of cells. Thatava et al were the first to successfully differentiate hiPSCs (human induced Pluripotent Stem Cells) into pancreatic β-cells in 2008. The group used hiPSCs from type 1 diabetes patients and the pancreatic β-cells they produced were able to secrete insulin in response to increased levels of glucose [81]. The differentiation of hiPSC derived pancreatic β-cells in humans however, still remains controversial due to variability in differentiation potential of these cells.

Mesenchymal stem cells from the human umbilical cord matrix (UCMSCs) are currently under investigation to treat diabetes [82]. UCMSCs lack or exhibit very low levels of cell surface markers that are detected by the immune system. This underlying ability to remain undetected by the immune system makes these cells ideal in the treatment of type 1 diabetes. They can also stimulate β-cells by secreting growth factors such as IGF1. Allogeneic UCMSCs have also been used to treat type 2 diabetes and were able to improve islet cell function in these patients [83]. This approach however is limited to patients who have their umbilical cord blood banked. Bone marrow derived mesenchymal stem cells have been used in such cases and have been proven to be safe and efficient in new onset type 1 diabetic patients [84]. An overview of the clinical trials being conducted using mesenchymal stem cells is listed in Table 3.6 [85]. A number of trials have proven the safety and efficacy of mesenchymal stem cells. The therapeutic benefit mediated by MSCs largely relies on the contribution of the secreted growth factors and cytokines rather than on their potential for differentiation to islets.

Hematopoietic stem cells (HSCs) have been tested in clinical trials for diabetes with the intent to re-educate the immune system and to halt autoimmunity toward β-cells [85]. Autologous hematopoietic stem cells transplantation (AHSCT) has also been used as a therapeutic option for new onset type 1 Diabetes in recent times [86]. The main strategy in this approach is to stop the pathogenic destruction of β-cells before all of them are destroyed. This is achieved by immunosuppressant drugs and is known as immunoablation. Immunoablation is followed by transplanting hematopoietic stem cells, which then regenerate and reshape the immune system thus making it more tolerant towards β-cells. This essentially resets the patient’s immune system [87].

 

Clinical Trial ID Phase Condition Outcome Measures Mode of Delivery Cell source
NCT01157403 2|3 T1D C-peptide IV Autologous MSC
NCT01759823 2|3 T2D eIns, C-peptide, IS IPA Autologous BM-MSC
NCT00690066 2 T1D C-peptide, eIns , HbA1c, Hypos, AA IV Allogeneic BM-MSC
NCT01219465 1|2 T1D C-peptide, eIns, BG; HbA1c, Hypos, Graft IV Allogeneic UC-MSC
NCT01374854 1|2 T1D C-peptide, Adv, FBG, eIns, HbA1c IPA Allogeneic UC-MSC, MNC
NCT00703599 1|2 T1D eIns, HbA1c, C-peptide, QoL, Adv IV Autologous AT-MSC
NCT01322789 1|2 T1D C-peptide, eIns, HbA1c, AA, Graft IV Allogeneic BM-MSC
NCT01496339 1|2 T1D HbA1c, Adv,Hypos, C-peptide, FBG, pBG, BG IPA/IV Allogeneic Men-MSCs
NCT00646724 1|2 T1D eIns, HbA1c, BG, C-peptide, Adv, Angio, AA, BC IPV Allogeneic Islets + MSC
NCT01413035 1|2 T2D Eff; Adv IV Allogeneic UC-MSC, PLA-MSC
NCT01453751 1|2 T2D eIns, Adv, eIns, HbA1c IPA/IV Autologous AT-MSC
NCT00703612 1|2 T2D BG, eIns, WB, HbA1C IV Autologous AT-MSC
NCT01576328 1|2 T2D Adv IV Allogeneic MPC
NCT01686139 1|2 CLI Adv, WH IM Allogeneic BM-MSC
NCT01257776 1|2 CLI Angio, Adv, ABI, WH IA AT-MSC
NCT01216865 1|2 DF, CLI Angio, Pain, ABI, WH, WD, Amp IM Allogeneic UC-MSC
NCT00955669 1 DF Angio IM Autologous MSC v/s MNC
NCT01068951 1 T1D C-peptide IV Autologous MSC
NCT01143168 1 T1D eIns, HbA1c, FBG, pBG, C-peptide, Adv IPA/IV Auto MNC + Autologous UC-MSC
NCT01142050 1 T2D IS, C-peptide, Ins, HbA1c, FBG, pBG, C-peptide, sIns, Adv IPA/IV BM-MSC
(not available) 1 T2D, DF Tregs/Th17/Th1, WH, CK, BG, eIns, ABI, TCpO2 IM Allogeneic UC-MSC
(not available) 1 T2D eIns, C-peptide, HbA1c, Adv, renal and cardiac function IV Allogeneic PLA-MSC
T1D: Type 1 Diabetes; T2D: Type 2 diabetes; CLI: Critical limb ischemia in T1D/T2D; DF: Diabetic foot; C-peptide: C peptide release; eIns: exogenous Insulin dose required; sIns: serum Insulin; BG: blood glucose; HbA1c: glycosylated Hemoglobin A1c; FBG: fasting blood glucose; Hypos: Number of severe and documented hypoglycemic events; Adv: safety and adverse events related to the procedure; pBG: Postprandial blood glucose; Graft: Immunologic reconstitution parameters; AA: levels of autoantibodies; Eff: Efficacy (in general); QoL: Quality of Life; IS: Insulin sensitivity; Angio: Angiographic assessment; ABI: Ankle Brachial Index; BC: Complete blood count; WH: Wound healing; WD: Walking distance; Amp: Rate and extent of amputations; Auto: Autologous Transplantation; Allo: Allogeneic Transplantation; MSC: Mesenchymal Stem Cells; UC-MSC: Umbilical cord-derived MSC; BM-MSC: Bone Marrow-derived MSC; PLA-MSC: Placenta-derived MSC; AT-MSC: Adipose Tissue-derived MSC or Adipose-Derived Stem Cells; Men-MSC: Menstrual Blood-derived MSCs; MPC: Mesenchymal Precursor Cells; MNC: Mononuclear cells; IV: Intravenous infusion; IPA: Intra-Pancreatic Artery infusion; IA: Intraarterial infusion; IPV: Intra-Portal Vein infusion; IM: intramuscular injection in tigh/shin/wound; CK: Pro/Anti-Inflammatory cytokines; Tx: Transplantation.

Table 3.6: Clinical trials involving MSCs for diabetes [85].

 

A study conducted by Li et al in 13 Chinese patients found that AHSCT was efficient in new onset type 1 diabetes. This observation is supported by other studies, which have been reported that the patients who received this therapy were no longer dependent on insulin administration for over 18 months. The mechanism behind this improvement has been credited to preservation of β-cell population [87]. This treatment has shown a greater efficacy in patients without diabetic ketoacidosis at diagnosis, indicating that the severity of the disease at the time of treatment could be predictive of the chances of preserving residual β-cells [88]. It is worthwhile to note that this therapy has considerable side effects by the drugs used for immunoablation and its application on a routine basis in its present form. It is fraught over [89]. The clinical trials being conducted on this front are listed in Table 3.7.

Several reports indicate that countries such as China and the Philippines remain at the forefront of regenerative medicine. These countries have been considered a safe haven for the advancements of incredible breakthroughs in the field of stem cells therapy due to their liberal ethical laws. There is an increasing trend of people travelling to these countries seeking cutting edge treatments thus popularizing stem cell tourism in Asia. The top destinations providing these treatments include Stem Care Institute, Manila, Philippines, ReLife International Medical Center, Beijing, China and 97.7 BnH Hospital, Stem Cell Treatment Center, Seoul, South Korea [90].

Xiumin Xu, director of China-USA Collaborative Human Cell Transplant Program at the Diabetes Research Institute at the University of Miami, Florida recently announced the results of a clinical trial that used mesenchymal stromal stem cells extracted from umbilical cord along with autologous bone marrow cell transplantation in 42 patients with type 1 diabetes [91]. The uniqueness of this study was that it was conducted in patients who had long standing type 1 diabetes. These patients were aged between 18 to 40 years. The combination of both cell types induce regeneration of pancreatic insulin secreting cells (β-cells), while the mesenchymal cells inhibited the T cell mediated immune response that was generated against the newly formed β-cells. The study demonstrated the safety of this approach as at 12 months, the patients in the transplanted group showed decreased symptoms of anxiety and depression and improved quality-of-life scores, 49% of the patients showed improved insulin secretion, while 71% showed significant increase in C-peptide secretion [92].

 

Clinical Trial ID Phase Condition Outcome Measures Route Cell source Results
NCT00315614 2 T1D beta cell function, Chimerism, Tolerance IV Autologous Islets and HSC Chimerism detected, but tolerance was not established
NCT00315133 1|2 T1D eIns, C-pep, HbA1c, QoL, AA, Graft IV Autologous HSC with ISp Insulin Independence achieved in patients with adequate residual beta cells
NCT00807651 2 T1D eIns, AA, C-pep, HbA1c IV Autologous HSC with ISp Insulin independence achieved in a subset of patients
NCT01341899 2 T1D C-pep, HbA1c, eIns, AA, Profile, Adv IV Autologous HSC with ISp Insulin independence in a subset of patients, reduced eIns requirements and HbA1c, increased C-pep
NCT00873925 2 T1D C-pep, DHA, VitD, HbA1c, eIns, Tcell IV Autologous UCB-HSC + VD + O3 Slowing of loss of endogenous insulin production
NCT00644241 2 T2D eIns, C-pep, HbA1c IV Autologous HSC Beta cell function improved, reduced eIns requirement
NCT00730561 N/A CLI, DN NCV IV Autologous HSC Improved perfusion, wound closure, no Amp
NCT01065337 2 DF Adv, Amp, WH, ABI, TCpO2, Per IV Autologous HSC v/s TRC Improved perfusion, wound closure
NCT00434616 2|3 PVD, DF, CLI Amp, WH, Pain, WD, QoL, TcpO2, ABI, Angio IV Autologous HSC Improved perfusion, no Amp
NCT00872326 1|2 PVD, DF Angio, ABI IV Autologous HSC Improved perfusion, vasculogenesis
NCT01065298 2|3 T2D eIns, C-pep, HbA1c IV Autologous HSC (not available)
NCT01694173 2|3 T2D C-pep, eIns, HbA1c, Cell Tracking IV Autologous HSC (not available)
NCT00971503 2 T1D C-pep, eIns, HbA1c, BG IV Autologous HSC (not available)
NCT01232673 2 CLI Amp, Per IV Autologous HSC (not available)
NCT01121029 1|2 T1D C-pep, Hb A1c IV Autologous HSC (not available)
NCT01285934 1|2 T1D C-peptide, eIns, HbA1c, C-pep IV Autologous HSC (not available)
NCT01786707 1|2 T2D HbA1c IV Autologous HSC + HOT (not available)
NCT01677013 1|2 T2D C-pep, HbA1c, Adv, Eff IV Autologous HSC (not available)
NCT00767260 1|2 T2D C-pep, Adv, HbA1c, eIns, FBG, sIns IV Autologous HSC + HOT (not available)
NCT00922389 1|2 DF, CLI Adv, Eff, TCpO2 IV Autologous HSC (not available)
NCT00465478 1|2 T1D, T2D eIns; HbA1c; BG, C-pep, Adv, Lip, AA, QoL IV Autologous HSC (not available)
NCT00788827 1 T1D, T2D, Kidney Tx Adv, Eff, QoL IV Autologous HSC (not available)
NCT01143168 1 T1D eIns, HbA1c, FBG, pBG, C-pep, Adv IV Autologous BM-MNC + Allogeneic UC-MSC (not available)
NCT00989547 1 T1D (not available) IV Autologous UCB-HSC (not available)
NCT00955669 1 DF Angio IV Autologous MSC v/s HSC (not available)
NCT01736059 1 Dry AMD, DR, RP Adv IV Autologous HSC (not available)
NCT00282685 1 DN DN, HR IV Autologous HSC (not available)
NCT01102699 4 T1D, T2D mCPC - mobilization (not available)
NCT00665145 2 T1D, T2D, DN Adv, Eff - mobilization (not available)
NCT01353937 1 DF WH,QoL, HbA1C, BG,TCpO2, ABI, Angio, Pain, Temp, Sen, GFR, DR - mobilization (not available)
T1D: Type 1 Diabetes; T2D: Type 2 diabetes; DN: Diabetic neuropathy; CLI: Critical limb ischemia in T1D/T2D; DF: Diabetic foot; PVD: Peripheral Vascular Disease; Dry AMD: Dry Age-related Macular Degeneration; RP: Retinitis pigmentosa; Kidney Tx: Kidney transplantation; C-pep: C peptide release; eIns: exogenous Insulin dose required; sIns: serum Insulin; BG: blood glucose; HbA1c: glycosylated Hemoglobin A1c; FBG: fasting blood glucose; Hypos: Number of severe and documented hypoglycemic events; Adv: safety and adverse events related to the procedure; pBG: Postprandial blood glucose; Graft: Immunologic reconstitution parameters; AA: levels of autoantibodies; Eff: Efficacy (in general); QoL: Quality of life; IS: Insulin sensitivity; Angio: Angiographic assessment; ABI: Ankle Brachial pressure Index; BC: Complete blood count; WH: Wound healing; TCpO2: Trans Cutaneous partial pressure of Oxygen on wound; WD: Walking distance; Amp: Rate and extent of amputations; Per: Tissue perfusion measurements; HSC: Hematopoietic Stem Cell; UCB-HSC: Umbilical Cord Blood HSC; BM-MNC: Bone Marrow Mononuclear Cells; IV: Intravenous infusion; IPA: Intra-Pancreatic Artery infusion; IA: Intraarterial infusion; IPV: Intra-Portal Vein infusion; IM: intramuscular injection in tigh/shin/wound; Profile: changes in lymphocyte immunophenotyping and cytokine profiles; NCV: nerve conduction velocity; Lip: Lipid profile; Temp: Temperature; Sen: Sensation; GFR: Glomerular filtration rate; DR: diabetic retinopathy; DHA: DHA Level; VitD: Vitamin D Level; Tcell: T-cell assays; HR: Heart rate; EPC: Endothelial Progenitor Cells; mCPC: mobilization of Circulating Progenitor Cell; Endo: Endothelial function; ISp: Immunosuppression; HOT: Hyperbaric Oxygen Therapy; VD: Vitamin D; O3: Omega3 fatty acids.

Table 3.7: Clinical trials involving HSCs for diabetes [89].

 

Cardion, Inc., in Erkrath, Germany is using embryonic stem cells to derive insulin producing human cells. The company uses a technique developed by Soria et al. This technique was able to eliminate cells that were not producing insulin from the culture, thus leaving behind only insulin producing cells. These cells were able to normalize glucose levels in experimentally induced diabetic mice [77]. The advantage of this approach lies in the fact that undifferentiated cells, which can develop into tumors are not transplanted into the patient.

The first clinical trial testing for safety and efficacy of stem cells for treatment of type 1 diabetes was initiated in July 2014 by ViaCyte a regenerative medicine company at San Diego, USA. The study used pancreatic progenitor cells derived from pluripotent stem cells, which were encapsulated in a protective coating that prevents their immunogenic destruction. The scientists hypothesized that the progenitor cells will spontaneously mature into islet cells in the pancreatic environment and secrete insulin, which will pass through the capsule and into the circulation. This approach has been successful in studies conducted in mouse models [93]. ViaCyte, Inc. also announced the opening of a second site in its Phase 1/2 trial called STEP ONE, or Safety, Tolerability and Efficacy of VC-01™ Combination Product in type 1 Diabetes, at the University of Alberta Hospital in Edmonton, Alberta funded in part by the Juvenile Diabetes Research Fund (JDRF) Canadian Clinical Trial Network (CCTN). The JDRF CCTN is an initiative to accelerate solutions for the management, care, and cure of type 1 diabetes. The JDRF CCTN creates a strong clinical research network to develop and conduct cutting-edge clinical trials in type 1 Diabetes and its complications, in order to accelerate delivery of the benefits of research advances to the community [94]. After closely monitoring the patients that received small doses of VC-01™, ViaCyte reported that the devices are working as expected without any side effects. Johnson & Johnson recently joined hands with ViaCyte to accelerate the development of this treatment that will eventually free type 1 diabetic patients from taking constant injections of insulin [95].

Timothy J. Kieffer, a Professor in molecular and cellular medicine at the University of British Columbia reported transplantation of human embryonic stem cell (hESC)-derived pancreatic progenitor cells, which were encapsulated in a protective membrane into immunosuppressed diabetic mice. The mice showed improvement in glucose tolerance but failed to attain normal levels of glucose. However oral intake of a low dose of a diabetic drug helped normalize the condition. Though preliminary, the study holds good prospects and scope for improvement. The approach that Kieffer used is similar to the one ViaCyte is using for their type 1 diabetes stem cell treatment [96].

A clinical trial in China is trying to assess the safety and efficacy of bone marrow stem cells for the treatment of type 2 diabetes. The study is investigating if injection of the patients´ own bone marrow stem cells into the pancreas can help treat this condition. The trial is being conducted at the 452 PLA Hospital in China's Sichuan Province and aims to recruit a total of 60 subjects. Another trial being conducted at the Peking University Aerospace Centre Hospital in Beijing, China is also evaluating the safety and effectiveness of using the patient's bone marrow stem cells in treating type 2 diabetes and expects to enroll about 500 patients [97].

A team at the Swiss Medica Clinic has reported reduction in insulin requirement by 80% in type 2 diabetic patients after administration of adipose derived mesenchymal stem cells over a period of six months. The treatment resulted in increased development of new blood vessels, secretion of various products of the immune system, and up regulation of pancreatic transcription factors as well as vascular growth factors. This stimulation created a favorable micro environment for β-cell activation and survival. This approach is said to be free of side effects and immune rejection and the stem cell reserve lasts for about 15 years thus rejuvenating the damaged pancreas in the process. No oncological event has been observed with this approach and it requires only a small quantity of fat tissue [98].

It is evident from the above mentioned overview that most clinical trials for the use of stem cells to cure diabetes are in Phase 1 or 2. We are still a few years away from these findings being fully and safely introduced into routine treatment. Nonetheless the prospects of this technology becoming available to the common man are not very distant. The fact that these studies are taking time to prove their safety and efficacy gives the general public assurance that these scientists are doing their best to ensure the safety of the human population and improve their quality of life at the same time.

 

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3.5 Stem Cells in the Treatment of Neurodegenerative Disorders

This chapter will begin with a brief description of the most important parts of the central nervous system (CNS) before dwelling on the main target diseases treatable by stem cell therapy [99]. The CNS is made up of the brain and spinal cord, which together form the command center of the nervous system. It communicates with the rest of the body through a complex and intricate network of neurons as shown in Fig. 3.10 nerve cells [99]. These nerve cells transmit information from all parts of the body to the brain and glial cells, which surround and support neurons.

 

TICEBA - The Story of Stem Cells - Chapter 3 - Fig. 3.10 - The neuron

Fig. 3.10: The neuron [99].

 

The central nervous system is known for its resistance to regeneration, which makes it a challenging field when it comes to treating of neurodegenerative disorders. Neurodegenerative disorders are a term used for a wide range of acute and chronic conditions, in which neurons and glial cells in the brain and the spinal cord are lost. For example, Parkinson’s disease is caused by the loss of cells that secrete dopamine, Amyotrophic lateral sclerosis (ALS) is caused by loss of motor neurons, and Alzheimer’s disease is caused by the death of cells that secrete neurotransmitters (chemicals with which nerve cells communicate), thereby hampering transmission of nerve impulses.

Generating fresh nerve cells based on stem cells offers a potential treatment for these diseases. The main approaches applied in stem cell therapy for neurodegenerative disorders are:

  1. By achieving stem cell-based nerve cell replacement and repair of the CNS. In this case, the transplanted stem cells differentiate into nerve cells and thereby help in reconstituting healthy nerve tissue.
  2. Stem cell therapy can also be used to modulate inflammation involved in the disease process thereby delaying the progress of the disease.
  3. Modulation of the host environment by paracrine factors that are released from transplanted stem cells. These factors can encourage the growth of stem cells present in the brain, thus letting the brain repair itself [100].

New research shows that transplanted stem cells migrate to the damaged areas and assume the function of neurons, holding out the promise of therapies for Alzheimer’s disease, Parkinson’s, Cerebral palsy, Spinal cord injury, Stroke, and other neurodegenerative diseases [101]. The etiology behind each of these diseases is discussed in detail below followed by the stem cell therapies being applied in their treatment.

Parkinson’s Disease
Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the progressive loss of neurons that secrete dopamine, which leads to impaired information processing in the brain. The main symptoms of PD are rigidity, poor movement, tremors, and postural instability. The pharmacological administration of L-dopa and dopamine receptor agonists has been found to be effective in treating some of the symptoms. However, the effectiveness of this approach decreases over time and is also associated with side effects [102]. Transplanting dopamine synthesizing cells is being considered as an alternative approach for restoring the damage. Human stem cells can serve as a source of cells for the treatment of PD [102]. Human embryo stem cells (ESCs) and human neural stem cells (NSCs) have been used for PD treatment in animal models. However, the post-transplantation survival of dopaminergic neurons generated from these cells has been poor. The number and survival rate of the stem cell-derived dopaminergic neurons need to be increased before making clinical applications possible [103]. Preclinical studies conducted in this direction that validate this approach are listed in Table 3.8.

 

Animal Model Transplanted Cells Additional Treatment Functional Outcome
Rat, 6-OHDA NPC (rat) FGF8/SHH Rotation (decreased)
Rat, 6-OHDA NSC (rat) - DA neuron None Not tested
Monkey, MPTP ESC (monkey) Stromal cell (mouse) feeder PFS-Parkinsonian factor score (decreased)
Rat, 6-OHDA Immortalized NSC (mouse, C17-2) TH/GTPCH1 Gene transfer Rotation (decreased)
Rat, 6-OHDA Immortalized NSC (human, HB1.F3) TH/GTPCH1 Gene transfer Rotation (decreased)
Rat, 6-OHDA Immortalized NSC (human, HB1.F3) NSC migration Rotation (decreased)
Monkey MPTP NSC (human) None PFS-Parkinsonian factor score (decreased)
Rat, 6-OHDA DA neurons from ES cells (human) None Rotation (decreased) Beam walking (increased)
Rat, 6-OHDA DA neurons from ES cells (human) Wnt signal Sonic hedgehog (Shh) Rotation (decreased)
Rat, 6-OHDA DA neurons from iPS cells (human) None Rotation (decreased)
6-OHDA: 6-hydroxydopamine; BMSC: bone marrow mesenchymal stem cell; ESC: embryonic stem cell; GTPCH-1: GTP cyclohydrolyrase-1; iPS cell: Induced pluripotent stem cell; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NPC: neural precursor cell; NSC: neural stem cell; TH: tyrosine hydroxylase [104].

Table 3.8: Stem cell-based cell therapy in experimental Parkinson's disease models.

 

Clinical trials of the transplantation of human fetal dopaminergic neurons have shown that cell replacement can produce major, long-lasting improvement in some patients, however for this approach to be transformed into a cure, a larger scale production of dopaminergic neurons needs to be established [102].

Huntington’s Disease
Huntington’s disease (HD) is a fatal, intractable disorder that is caused by the accumulation of aggregated forms of the huntingtin protein, which results in neuronal dysfunction and degeneration. This, in turn, contributes to the progressive physiological, motor, cognitive, and emotional disturbances characteristic for HD. The recent treatment of HD has centered on stem cell therapy strategies that are aimed to protect vulnerable neuronal cell populations or to replace dysfunctional or dying cells. At this time, using stem cells for the delivery of trophic factors and neuroprotection to prevent disease progression seems a more achievable clinical goal in HD than neuronal replacement [102]. Human NSCs derived from ESCs could provide a viable cellular source for cell therapy in HD, since they can be expanded indefinitely and differentiate into any cell type desired. Studies have shown that ESC-derived neurons in rats lead to behavioral recovery in the animals [104]. A summary of clinical trials of stem cell transplantation in HD is shown in Table 3.9.

 

Study (year) Clinical Size Type of Cell Clinical Outcome Negative Effects
Bachoud-Levi (2006, 2009, 2000) Five patients Whole ganglionic eminence Three of five patients showed stability of symptoms or clinical improvement for 4–6 years One patient showed development of a putaminial cyst
Capetian et al. (2009) One patient Whole ganglionic eminence UHDRS score stability for 6 months. Survival and differentiation of grafted cells None reported (patient died from unrelated causes)
Cicchetti et al. (2009, 2014) Three patients Lateral ventricular eminence containing striatal primordia Improvement of UHDRS in two of three patients for up to 18 months before returning to presurgical levels Grafts underwent disease-like neuronal degeneration. Cortical hemorrhage, subdural hematoma following surgery
Freeman et al. (2000) One patient Lateral ventricular eminence containing striatal primordia Stability of UHDRS 15 months following transplantation. Transplants integrated into the host tissue None reported
Furtado et al. (2005) Seven patients Fetal striatal tissue Transplants failed to restore fluorodeoxyglucose uptake and D1 and D2 receptor binding in subjects Possible technical issues with regards to the ganglionic eminence and in targeting the striatum
Hauser et al. (2002) Seven patients Fetal striata Grafts developed striatal morphology, UHDRS improved significantly 12 months following surgery Three subjects developed subdural hemorrhages, one patient died 18 months following surgery from probable cardiac arrhythmia
Keene et al. (2007) Two patients Fetal lateral ganglionic eminence Improved ambulation 3 months following transplant in one patient. In both patients, transplanted cells displayed morphology of neurons and astrocytes One patient reported chronic headaches following surgery and was treated for bilateral subdural hematomas. Reported that transplants did not have an effect on the course of HD
Keene et al.(2009) One patient Fetal neuronal tissue Clinical improvement for UHDRS for 2 years. Patient died 121 months following surgery from complications of advanced HD Three mass lesions and one large cyst were present on the left caudate and putamen. Five mass lesions and two cysts were present on the right caudate and putamen
Kopyov et al. (1998) Three patients Lateral ganglionic eminence Clinical improvement for UHDRS for all three patients 12 months following surgery. Graft survival and growth within the striatum without displacing host tissue None reported
Krystkowiak et al. (2007) 13 patients Fetal neuronal tissue Pre- and post-UHDRS were not reported. Four of the 13 patients had grafts that did not display signs of rejection Biological, radiological and clinical rejection of grafts in other subjects (reversible under immunosuppressive treatment)
Reuter et al. (2008) Two patients Whole ganglionic eminence Clinical improvement for UHDRS over 5-year period for one patient. Increased striatal D2 receptor binding, suggesting long-term survival and efficacy of grafts None reported
Rosser et al. (2002) Four patients Whole ganglionic eminence Stability of UHDRS as well as cognitive ability up to 6 months following surgery. Graft survival without overgrowth None reported
Philpott et al. (1997) Three patients Lateral ganglionic eminence Increased cognitive functioning 6 months following surgery None reported
Gallina et al. (2010) Four patients Whole ganglionic eminence Stability or improvement in motor, behavioral and functional scores up to 24 months following surgery None reported
Madrazo et al. (1995) Two patients Whole ganglionic eminence Stability or improvement on functional capacity for up to 25 months following surgery when a slow progression of HD was observed None reported

Table 3.9: Clinical trials on transplantation of stem cells in Huntington's disease. HD: Huntingdon's disease; UHDRS: Unified Huntington's disease rating scale [105].

 

Alzheimer’s Disease
Alzheimer’s disease (AD) is characterized by neuronal and synaptic loss throughout the brain. Although in AD massive neuronal loss only occurs in very few brain structures, large parts of the brain are affected by pathological alterations and decreased neuronal metabolism. These pathological changes seen in AD offer an extremely problematic situation for cell replacement. Current therapies, such as treatment with acetylcholinesterase inhibitors to enhance cholinergic function, provide only partial and temporary alleviation of symptoms. The data show that NCSs release diffusible factors that may improve the survival of aged and degenerating neurons in human brains [102]. MSCs from the umbilical cord have been used in the treatment of AD. A few of the ongoing clinical trials in this direction is described in Table 3.10.

 

Trial Name Stem Cell Type Transplant Type Delivery Method Trial Status
NEUROSTEM-AD/ Medipost Co Ldt. http://medi-post.com/ MSC from umbilical cord blood Allogenic Intracerebral implantation Phase 1 NCT01297218
NEUROSTEM-AD/ Duk Lyul Na* MSC from umbilical cord blood Allogenic Intracerebral implantation Recruiting NCT01696591
Affiliated Hospital to academy of military medical Science* MSC from umbilical cord blood Allogenic Intravenous Phase 1/2 NCT01547689
Ns Gene A/S* http://nsgene.dk/ Encapsulated cell biodelivery device: NsG0202 Allogenic i.c. (implantation into the basal forebrain nuclei) Phase 1 NCT01163825

Table 3.10: Stem cells in current clinical trials for Alzheimer’s disease [100].

 

Amyotrophic Lateral Sclerosis
Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease caused by progressive degeneration of motor neurons in the cerebral cortex, the brainstem, and the spinal cord and results in fatal paralysis. The survival of patients is limited to just 3 – 5 years after the onset of this disease [106]. The etiology behind ALS still remains unknown with about 90-95% of cases being sporadic in nature. The heterogeneity in symptoms of ALS makes it difficult to identify the mechanism and origin of the disease thus hampering the process of developing a potent cure [107]. A stem cell-based approach holds great promise for the treatment of ALS. The biggest challenge for the treatment to be effective is that the transplanted stem cells need to survive and form long-distance projections from the brain to the spinal cord and/or spinal cord to muscle, with functional connections. Adding to this situation is the fact that the new cells are transplanted into an already toxic environment amidst neurons that are already degenerating, which makes the survival of the transplanted cells harder. An alternative approach is also being considered for creating a microenvironment that aids in reconstructing the damaged area. This can be achieved by transplanting supporting cell types that will release growth factors and help detoxification the damaged area, thereby supporting the survival of existing motor neurons [107].

MSCs derived from the bone marrow and NSCs derived from the fetal brain have been used to generate immunomodulatory cells, growth factor-releasing cells, functional support cells such as glia, or GABAergic interneurons to modify motor neuron survival and activity in an attempt to rescue existing motor neurons [108]. MSCs are ideal for stem cell therapy as they avoid ethical restrictions and provide the possibility of autologous transplantation. Mazzini and colleagues were the first to perform clinical studies to determine the safety and tolerability of directly transplanted MSCs (or any cell type) to treat ALS [107]. A number of other clinical trials have also been conducted in this direction as shown in Table 3.11.

 

Trial Name Stem Cell Type Cell Specifics Delivery Method Trial Status Pre-clinical Rationale for Clinical Translation
Mazzini MSC Autologous, derived from patient bone marrow Spinal cord injections Phase I complete, No current studies N/A
Brainstorm MSC Autologous, derived from patient bone marrow Intrathecal, intramuscular Phase I complete, Phase II ongoing In vivo secretion of neurotrophic factors, beneficial effects in Parkinson's and Huntington's disease rodent models
Martinez Stem cells Autologous, derived from patient blood Frontal cortex injections N/A N/A
Neural stem NSC Human fetal spinal cord (at 8 weeks) Spinal cord injections Phase I complete, Phase II current Enhanced survival of spinal motor neurons in rats
Q Therapeutics GRP Human fetal forebrain (at 17–24 weeks) Spinal cord injections Preclinical Beneficial effects on motor function, lifespan and spinal motor neurons, decreased microgliosis in rats; using rat GRPs
Cedars-Sinai hNPC releasing GDNF Human fetal cortex (at 8–15 weeks) Spinal cord injections Preclinical GDNF secretion in vivo enhances survival of spinal motor neurons in rats
Vescovi NSC Human fetal (unspecified) Spinal cord injections Phase I current N/A

Table 3.11: Summary of stem-cell-based clinical trials for ALS [107].

 

Stem Cell Therapy in Stroke
Stroke is a sudden death of brain cells in a particular area caused by an interrupted blood flow. There are two types of stroke: a) ischemic stroke, caused by an occlusion of a blood vessel, and b) Hemorrhagic stroke, initiated by a rupture of a blood vessel in the brain. Most of the stroke cases are ischemic (85-90%) as compared to hemorrhagic (10-15%). Currently, the only accepted treatment for stroke is the administration of antithrombotic agents, but their use is often limited by a narrow therapeutic time window [102]. The application of stem cells in the treatment of stroke has been pursued with a hope to find a cure. MSCs are one of the most commonly used types of stem cell in clinical trials on stroke to date [109]. Preclinical studies have been performed on rats with induced spinal cord injury and have shown that transplantation of MSCs derived from bone marrow and adipose tissues are not only able to survive, but to migrate into the host tissue and lead to axonal regeneration and motor function recovery [102]. Clinical trials being conducted with this objective are listed in Table 3.12 [100].

 

Trial No & Status Stage of Stroke Stem Cell Type Transplant Type Delivery Method Name/Sponsor
Phase 2 NCT00950521 CS HSC (CD34+ cells) from peripheral blood Autologous i.c. China Medical University Hospital*
NCT01239602 CS HSC (CD34+ cells) from peripheral blood Autologous i.c. China Medical University Hospital*
Phase 1 NCT01518231 CS HSC (CD34+ cells) from peripheral blood Autologous i.a. AHSCTIS/ Zhejiang Hospital*
Phase 1 NCT01438593 CS HSC (CD34+ cells) from umbilical cord blood Allogenic i.c. China Medical University Hospital*
Phase 1/2 NCT00761982 AS HSC (CD34+ cells) from bone marrow Autologous i.a. Hospital Universitario Central de Asturias*
Phase 2 NCT01501773 AS HSC (mononuclear cells) from bone marrow Autologous i.v. Manipal Acunova Ltd.*
Phase 1 NCT00859014 AS HSC (mononuclear cells) from bone marrow Autologous i.v. University of Texas Health Science Center*
Phase 1 NCT00473057 AS/SAS HSC (mononuclear cells) from bone marrow Autologous i.v. Federal University of Rio de Janeiro*
Phase 1/2 Bang et al., 2005 AS MSC from bone marrow Autologous i.v. STARTING/ Korea Health 21 R&D Project
Phase 3 NCT01716481 AS MSC from bone marrow Autologous i.v. STARTING-2/ Samsung Medical Center*
Lee et al., 2010 AS MSC from bone marrow Autologous i.v. Korea Research Foundation and the Korea Health 21 R&D Project
Phase 2 NCT01461720 AS MSC from bone marrow Autologous i.v. National University of Malaysia*
Phase 2 NCT00875654 SAS MSC from bone marrow Autologous i.v. ISIS / University Hospital, Grenoble*
Phase 1/2 NCT01468064 AS MSC from bone marrow Autologous i.v. AMETIS/ Southern Medical University, China*
Phase 1/2 NCT01297413 CS MSC from bone marrow Autologous i.v. Stemedica Cell Technologies, Inc.*
Phase 1/2 NCT01453829 S MSC from adipose tissue Autologous i.v./i.a. Ageless Regenerative Institute*
Phase 2 NCT01389453 SAS MSC from umbilical cord blood Allogenic i.v. General Hospital of Chinese Armed Police Forces*
Phase 1 NCT01151124 CS NSC (CTX0E03) Allogenic i.c. PISCES/ ReNeuron Limited*
Phase 1 NCT01327768 CS hOEC Autologous i.c. OECs/ China Medical University Hospital*
Stages of stroke: S: stroke; AS: acute ischemic stroke; SAS: subacute ischemic stroke; CS: chronic ischemic stroke; Administration routes: i.a.: intra-arterial; i.v.: intravenous; i.c.: intracerebral implantation; Types of stem cells: HSC: Hematopoietic Stem Cells; NSC: Neural Stem Cells; MSC Mesenchymal Stem Cells; OEC: Olfactory Ensheathing Cells [98].

Table 3.12: Stem cells in current clinical trials for ischemic stroke [100].

 

In 2006, a research group from Germany demonstrated that NSCs were able to give rise to new nerve cells inside the brain and the neurons they produced could also make connections to existing neurons of the brain. Several other research groups have demonstrated that transplanted neurons produced from human embryonic stem cells were able to integrate into rat brains after they had undergone an ischemic stroke. The scientists observed an improvement in the movement of the animals after the transplant. A recent study led by groups from Sweden and Germany has revealed similar results in mice and rats using NSCs made from human iPS cells. These results, however, are mainly from preclinical studies in animal models and scientists need to understand precisely how to guide the pluripotent stem cells to produce only the type of neural cell required to produce methods for transplantation that will be safe and effective [109].

Thailand is considered as one among the leaders in the application of stem cell therapy and has superior stem cell clinics for the treatment of neurodegenerative diseases such as Parkinson’s diseases, diabetes, Alzheimer’s disease, types of arthritis and much more. [110] Beike Biotechnology is partnered with the Better Being Hospital (BBH) in Bangkok, Thailand, to provide the most extensive stem cell-based treatment to a number of diseases, including neurodegenerative diseases. The Cellular Therapeutics Center of Makati Medical Center, Philippines is equipped with world-class instruments from Germany, Japan, and the USA. The lab offers a wide range of services that boast remarkable efficacy of stem cells in treating a number of diseases. This is a Clean Room ISO Class V facility, which exceeds the recommendations of the US-FDA (United States Food and Drug Administration). As part of the routine procedure, the facility is continuously monitored for its sterility. Clinicians and scientists at this center are looking at the potential use of stem cells to treat a number of conditions, including Parkinson's disease, Alzheimer's disease, cancer, spinal cord injury, heart disease, diabetes, and arthritis. This center only uses autologous cells to prevent complications and transplant rejection [111].

A group of Chinese scientists recently generated a type of nerve cell that is typically lost (or dysfunctional) in the brains of AD patients and some mouse models of AD from human embryonic stem cells. When the cells were transplanted into the basal forebrain of AD mice, most of the cells survived and matured into adult cholinergic nerve cells that were able to function in tandem with the original mouse nerve cells. The study concluded that replacing cholinergic nerve cells in the basal forebrain area of the brain is a potential approach to reversing memory loss in Alzheimer’s disease. The lead in this study, Dr. Naihe Jing cautioned that the results need to be reproduced in primate models before being tested for human trials [112].

The sensitivity and critical nature of neurodegenerative diseases make them an extremely challenging set of diseases to cure. Though a complete cure is a little farsighted, stem cells appear to aid in the improvement of the quality of life of these patients. Compared to other diseases, stem cell therapy for neurodegenerative diseases appears to be only at the initial stages of clinical trials being conducted in a limited number of patients. A number of articles online seem to caution patients to adequately investigate the center prior to accepting stem cell therapy for these diseases.

 

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3.6 Stem Cells in Wound Healing

Any injury to the human body triggers an organized and complex cascade of molecular and biochemical events that initiate the healing of the damaged area [113]. This chapter will address one of the most promising applications of stem cells - wound healing. The term wound has been defined as a disruption of a normal anatomical structure and, more importantly, function [114]. This can range from a simple break in the epithelial integrity of the skin or it can be deeper, extending into subcutaneous tissue with damage to other structures such as tendons, muscles, vessels, nerves, parenchymal organs, and even bone. Wounds can be clinically categorized as acute and chronic according to their time frame of healing. Wounds that repair themselves and that proceed normally by following a timely and orderly healing pathway, with the end result of both functional and anatomical restoration, are classified as acute wounds. The time course of healing usually ranges from 5 to 10 days, or within 30 days. Chronic wounds are those that fail to progress through the normal stages of healing and they cannot be repaired in an orderly and timely manner. The healing process is incomplete and disturbed by various factors, which prolong one or more stages in the phases of wound healing. These factors include infection, tissue hypoxia (lack of oxygen supply to the tissue), necrosis (cell death), exudates, and excess levels of inflammatory cytokines. Chronic wounds may result from various causes, including pressure, arterial and venous insufficiency, burns, and vasculitis [115].

To gain a better insight into the pathological conditions associated with compromised wound healing we will first go through the response of normal tissue to injury. There are four possible responses to an injury incurred to the human body.

  1. Normal repair is the response where there is a re-established equilibrium between scar formation and scar remodeling. This is the typical response of the human body to injury.
  2. Regeneration is the process that occurs when the injured structure is replaced exactly to what was there before the injury. For example, lower forms of life, such as the salamander and crab, can regenerate tissues. The human body has restricted regenerative capabilities, however, the liver, epidermis and, to some extent, nerves can be partially regenerated after injury.
  3. In excessive healing, there is excessive deposition of connective tissue that results in altered structure and, thus, loss of function. Fibrosis, strictures, adhesions, and contractures are examples of excessive healing. Contraction is part of the normal process of healing but if excessive, it becomes pathologic and is known as a contracture.
  4. Deficient healing is the opposite of fibrosis; it exists when there is insufficient deposition of connective tissue matrix and the tissue is weakened to the point where it can fall apart. Chronic nonhealing ulcers are examples of deficient healing [116].

 

TICEBA - The Story of Stem Cells - Chapter 3 - Fig. 3.11 - Responses to Tissue Injury

Fig. 3.11: Responses to Tissue Injury.

 

Normal wound healing is a dynamic and multistep process involving coordinated interactions between diverse immunological and biological systems that result in the restoration of anatomical continuity and function [117]. Before we dive into the phases of wound healing, let us familiarize with basic components involved in this process. Platelets and inflammatory cells are the first cells to arrive at the site of injury and they provide "signals" needed for the influx of connective tissue cells and the generation of a new blood supply. These chemical signals are known as cytokines or growth factors. The fibroblast is the connective tissue cell responsible for collagen deposition that is needed to repair the tissue injury. Collagen is the most abundant protein in the animal kingdom, accounting for 30% of the total protein in the human body. In normal tissues, collagen provides strength, integrity, and structure. When tissues are disrupted following injury, collagen is needed to repair the defect and restore anatomic structure and function [116].

Wound healing comprises of four overlapping phases of coagulation or hemostasis, inflammation, migration-proliferation (including matrix deposition), and remodeling. These mechanisms are initiated at the time of physical injury and proceed continuously throughout the repair process. All these steps initiate a coordinated and sequential movement of specialized cells into the injured site to facilitate its recovery.

The phases involved in normal wound healing are discussed in detail below.

Initial Insult and Inflammation
Wound healing is initiated immediately after an injury and begins with the activation of the coagulation cascade. The coagulation cascade gets activated when platelets come in contact with exposed collagen at the site of injury. Platelets begin to accumulate at this site and begin to release clotting factors, which result in the formation of a fibrin clot. The blood clot re-establishes hemostasis and provides a provisional extracellular matrix (ECM) for cell migration. Apart from the release of clotting factors, platelets also release a cascade of chemical signals, known as cytokines or growth factors that initiate the healing response. The two most important signals are platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-β). PGDF initiates chemotaxis of neutrophils, macrophages (the immune cells), and fibroblasts to the site of injury to prevent infection. This process is known as inflammation (Fig 3.12). Inflammation is the key process, which can determine not only the rate of healing but also the degree of fibrosis. Excessive inflammatory reactions have been shown to result in delayed or incomplete healing and an increase in the severity of fibrosis.

 

TICEBA - The Story of Stem Cells - Chapter 3 - Fig. 3.12 - Phase 1 of wound healing

Fig. 3.12: Phase 1 of Wound Healing: Inflammation. Growth factors thought to be necessary for cell movement into the wound are shown. TGF-β1, TGF-β2, and TGF-β3 denote transforming growth factor β1, β2, and β3, respectively; TGF-α transforming growth factor α; FGF fibroblast growth factor; VEGF vascular endothelial growth factor; PDGF, PDGF-AB, and PDGF-BB platelet-derived growth factor, platelet-derived growth factor AB, and platelet-derived growth factor-BB, respectively; IGF insulin-like growth factor; and KGF keratinocyte growth factor [118,119].

 

The first immune cells to migrate to the wound site are neutrophils in response to PDGF and transforming growth factor-beta (TGF-β). Neutrophils are the predominant cell marker in the wound site within 24 hours after injury. The major function of neutrophils is to remove foreign material, bacteria, non-functional host cells, and damaged matrix components that may be present at the wound site. The phagocytotic activity of neutrophils is crucial for the subsequent processes because acute wounds that have a bacterial imbalance will not heal. Neutrophils will consume all bacteria and debris until they are filled and constitute what is called "pus" in the wound. The classic signs of inflammation include rubor (redness), calor (heat), tumor (swelling), and dolor (pain) [116].

Neutrophil activity gradually changes within a few days, once all the contaminating bacteria have been removed. Upon completing the task, the neutrophils must be eliminated from the wound prior to progression to the next phase of healing. Redundant cells are disposed of by extrusion to the wound surface as slough and by apoptosis, allowing elimination of the entire neutrophil population without tissue damage or potentiating the inflammatory response. The cell remnants and apoptotic bodies are then phagocytosed by macrophages [115].

By 48 hours after injury, fixed tissue monocytes become activated to become wound macrophages and move into the site of injury and transform into very active wound macrophages. These highly phagocytic cells also release PDGF and TGF-β to recruit fibroblasts to the site [116]. Macrophages have a longer lifespan than neutrophils and continue to work at a lower pH.

Matrix Formation
As the inflammatory phase of wound healing is toned down wound contraction begins. Matrix formation begins around 72 hours after wounding and is facilitated by fibroblasts and is also known as the proliferative phase. This phase is characterized by fibroblast migration and deposition of the newly synthesized extracellular matrix, acting as a replacement for the provisional network composed of fibrin and fibronectin. Formation of ECM proteins, angiogenesis, contraction, and keratinocyte migration are essential components of these phases. Matrix proteins, including collagens, fibronectin, and vitronectin provide substrates for cell movement, vehicles for changing cell behavior, and structures that return function and integrity to the tissue [119]. At the macroscopic level, this phase of wound healing can be seen as an abundant formation of granulation tissue (Fig 3.13). New stroma, often called granulation tissue, begins to invade the wound space approximately four days after injury. Macrophages, proliferating fibroblasts, and vascularized stroma, together with a collagen matrix, fibrinogen, fibronectin, and hyaluronic acid constitutes the acute granulation tissue that replaces the fibrin-based provisional matrix [115].

PDGF and TGF-β attract fibroblasts to the wound site. Once within the wound the fibroblasts proliferate profusely and produce the matrix proteins hyaluronan, fibronectin, proteoglycans, and type 1 and type 3 procollagen. Collagens are an important component in all phases of wound healing. Synthesized by fibroblasts, they impart integrity and strength to all tissues and play a key role, especially in the proliferative and remodeling phases of repair. By the end of the first week, abundant extracellular matrix accumulates, which further supports cell migration and is essential for the repair process.

Due to the high metabolic activity at the wound site, there is an increasing demand for oxygen and nutrients. Local factors in the wound microenvironment such as low pH, reduced oxygen tension and increased lactate actually initiate the release of factors needed to bring in a new blood supply [116]. Angiogenesis or neovascularization, the process of forming blood vessels throughout the injured skin, also occurs around this phase (Fig 3.13). Angiogenesis enables the re-supply of oxygen and other nutrients. The establishment of new blood vessels is critical in wound healing and takes place concurrently during all phases of the reparative process. A blood supply is required to supply the injured skin with nutrients and oxygen to enable cellular migration, proliferation, and differentiation. This enables the endothelial cells to proliferate and migrate into the wound site to form a new blood vessel network [118].

Contraction, aided by the formation of ECM, granulation tissue, and the emergence of myofibroblasts, is a rapid and efficient way of achieving wound closure.

 

TICEBA - The Story of Stem Cells - Chapter 3 - Fig. 3.13 - Phase 2 of Wound Healing

Fig. 3.13: Phase 2 of Wound Healing: Angiogenesis and reepithelialization of wounds. Proteinases thought to be necessary for cell movement are shown. The abbreviation u-PA denotes urokinase-type plasminogen activator; MMP-1, 2, 3, and 13 matrix metalloproteinases 1, 2, 3, and 13 (collagenase 1, gelatinase A, stromelysin 1, and collagenase 3, respectively); and t-PA tissue plasminogen activator [118].

 

Remodelling
This phase is the final phase in wound healing and includes events such as collagen synthesis, degradation as well as reorganization, and often the formation of scar tissue. There is also the gradual replacement of collagen III with collagen I. Synthesis and breakdown of collagen as well as extracellular matrix remodeling take place continuously and both tend to equilibrate to a steady state about three weeks after injury. Matrix metalloproteinase enzymes, produced by neutrophils, macrophages, and fibroblasts in the wound, are responsible for the degradation of collagen. Their activity is tightly regulated and synchronized by inhibitory factors. Gradually, the activity of tissue inhibitors of metalloproteinases increases, culminating in a drop in activity of metalloproteinase enzymes, thereby promoting new matrix accumulation [115].

Although the initial deposition of collagen bundles is highly disorganized, the new collagen matrix becomes more oriented and cross-linked over time. Its subsequent organization is achieved during the final stages of the remodeling phase, to a greater extent by the wound contraction that has already begun in the proliferative phase. Due to fibroblast interactions with the extracellular matrix, underlying connective tissue shrinks in size and brings the wound margins closer together. The process is regulated by a number of factors, most important among them being PDGF, TGF-β, and FGF. As the wound heals the density of fibroblasts and macrophages is further reduced by apoptosis. With time, the growth of capillaries stops blood flow to the area declines and metabolic activity at the wound site decreases. The end result is a fully matured scar with a decreased number of cells and blood vessels and a high tensile strength. Multiple factors can lead to impaired wound healing. They can be broadly classified into systemic and local factors. These factors may slow the course of wound healing by causing disturbances in the finely balanced repair processes, resulting in chronic, nonhealing wounds (Table 3.13).

Local Factors Systemic Factors
Oxygenation Infection Foreign body* Venous sufficiency Age and gender Sex hormones Stress Ischemia Diseases: diabetes, keloids, fibrosis, hereditary healing disorders, jaundice, uremia Obesity Medications: glucocorticoid steroids, non-steroidal anti-inflammatory drugs, chemotherapy Alcoholism and smoking Immunocompromised conditions: cancer, radiation therapy, AIDS Nutrition

Table 3.13: Factors Affecting Wound Healing [120]. *Oxygenation as in lack of oxygen supply to the site of the wound, Infection as in any secondary infection to an existing wound, Foreign bodies as in debris that deposit on wounds, Venous sufficiency as in the case of decreased blood supply to the site of the wound. Table 3.13 shows a list of factors that affect the process of wound healing.

 

Local factors that affect wound healing

Oxygenation

The availability of oxygen is crucial for normal cell metabolism as it drives the production of ATP, which is the energy currency of the cell. Optimal oxygen levels play a critical role in the wound healing process. Oxygenation directly impacts the amount of ATP available at the site of the wound to drive all stages during the wound healing response. Adequate oxygenation prevents wounds from infection, induces angiogenesis, enhances fibroblast proliferation, increases keratinocyte differentiation, migration, and re-epithelialization, and collagen synthesis, and promotes wound contraction. The wound site is usually deprived of optimal oxygenation due to the disruption of blood supply and excessive oxygen uptake by metabolically active cells. This creates a hypoxic microenvironment at the wound site. Prolonged hypoxia is also known to hamper wound healing. Chronic wounds are known to be hypoxic [121].

Venous sufficiency
Venous sufficiency is intricately associated with oxygenation. Sufficient vasculature at the wound site becomes a crucial factor that affects the quality and the duration of time is taken for the wound to heal. The high amount of activity at such sites demands active metabolism and energy production. Wound site, however, is inevitably associated with disrupted vasculature thereby hampering the healing process. Vascular insufficiency at the wound site can severely impact wound healing as the proper blood supply is the only mode to supply nutrients in the body. This is commonly observed in patients suffering from chronic venous insufficiency. These patients suffer from chronic wounds. Restoration of venous sufficiency by stem cell-based approach has been gaining popularity lately. This approach will be discussed separately under chronic venous insufficiency.

Infection
Open wounds are often susceptible to secondary infections with species like P. aeruginosa and Staphylococcus that are common in bacterial infection of wounds. The presence of biofilms containing P. aeruginosa in chronic ulcers causes significant hindrance in the wound healing process. If untreated, infections can progress to sepsis, osteomyelitis, and gangrene. Another factor that affects the healing process is the presence of foreign bodies or debris, such as necrotic tissue.

Foreign Bodies
Foreign material includes sutures; dressing residues; fibers shed by dressings; and foreign material introduced during the wounding process, such as dirt or glass. These particles must be removed from the wound site in order to allow the wound to progress from the inflammatory stage to the proliferative stage of wound healing [122].

Systemic factors that affect wound healing
The systemic factors that affect wound healing include age, hormone levels, nutritional status and overall health of the individual. Aging is associated with thinning of the epidermis making the fragile and highly susceptible to injury. Moreover, the decreased metabolic rates in aged people further reduce the rate of healing. Co-morbidities such as diabetes, keloids, fibrosis, hereditary healing disorders, jaundice, uremia, obesity, cardiopulmonary diseases etc. result in poor tissue perfusion of nutrients thereby limiting cellular activity. Co-morbidities such as cancer, radiation therapy, AIDS etc. suppress or compromise the immune response and hamper the inflammation process during wound healing. A compromised immune system also renders the wound vulnerable to secondary infections from microorganisms [122]. Medications that interfere with clot formation or platelet function, or inflammatory responses and cell proliferation have the capacity to affect wound healing. Anti-inflammatory drugs, immunosuppressives, anticoagulants, antineoplastic agents, steroid and oral contraceptive agents can impede the wound healing process and cause undesirable physiologic effects such as poor or prolonged inflammatory response, reduced blood supply, delayed collagen synthesis, and decreased tensile strength of repaired tissue [120].

Lifestyle factors also play an important role in the wound healing process. Good nutrition and regular physical exercise enhance tissue perfusion and thus improves the availability of nutrients needed to sustain the increased metabolic activity at the wound healing site. A diet rich in antioxidants and minerals will aid in wound healing. Protein deficiency can impair capillary formation, fibroblast proliferation, proteoglycan synthesis, collagen synthesis, and wound remodeling. Vitamin C is required for collagen synthesis, fibroblast functions, and the immune response. Vitamin A aids macrophage mobility and epithelialization. The biological properties of vitamin A include anti-oxidant activity, increased fibroblast proliferation, modulation of cellular differentiation and proliferation, increased collagen and hyaluronate synthesis. Vitamin B complex is necessary for the formation of antibodies and WBCs, and Vitamin B or thiamine maintains metabolic pathways that generate the energy required for cell reproduction and migration during granulation and epithelialization. Iron is required for the synthesis of hemoglobin, which carries oxygen to the tissues, and copper and zinc play a role in collagen synthesis and epithelialization [122]. Vitamin E, an antioxidant, maintains and stabilizes cellular membrane integrity by providing protection against destruction by oxidation. Vitamin E also has anti-inflammatory properties and has been suggested to have a role in decreasing excess scar formation in chronic wounds. Several micronutrients such as Magnesium, copper, and zinc have been shown to be important for optimal repair of wounds.

Alcohol abuse and smoking also interfere with body’s defense system and thus hamper wound healing. Clinical studies and animal experiments have provided evidence that exposure to alcohol consumption impairs wound healing and increases the incidence of infection [123,124]. Patients who smoke show a delay in wound healing and an increased susceptibility to infection, wound rupture, anastomotic leakage, wound and flap necrosis, epidermolysis, and a decrease in the tensile strength of wounds [125,126].

The body has limited healing capabilities and this process is often flawed as accelerated healing is preferred, with the new tissue being architecturally distinct from the original and is also accompanied by loss of function or pain. This results in scar formation. When wounds are beyond the repair capability of the body, their healing remains incomplete thereby resulting in chronic wounds. This results in scar formation. When wounds are beyond the repair capability of the body, their healing remains incomplete thereby resulting in chronic wounds. Wounds that are beyond the capability of the body to repair them remain incomplete resulting in chronic wounds.

 

Chronic Wound Healing by Stem Cell Therapy

Wounds are becoming an increasing clinical burden due to the high incidence of diabetes, obesity, and an aging population [127]. Therefore, scientists are on the lookout to speed up and improve the efficacy of the healing process by the application of stem cells. Stem cell-based therapies hold great potential on this front. Chronic wounds are rarely seen in otherwise healthy individuals; they are often associated with diabetes, obesity or old age. It has been estimated that 1-2% of people in developed countries suffer from chronic wounds in their lifetime [128]. Current methods of wound management are palliative, but their ineffectiveness for complex wounds is an ongoing clinical problem. Healthcare systems are in desperate need of alternative therapies. Stem cells are known to tremendously influence normal cell and tissue repair/regeneration, which is why a large proportion of research focuses on stem cells as the answer to treating chronic wounds. Self-renewing characteristics and multipotent differentiation potential of stem cells make them ideal candidates for the treatment of chronic wounds. Stem cell-based therapies bring about their effectiveness via a number of mechanisms. Stem cells can differentiate into new cells, secrete trophic factors, promote angiogenesis, modulate the immune system, improve wound closure, and also help in the development of new extracellular matrix (ECM) (Fig 3.14) [127].

 

TICEBA - The Story of Stem Cells - Chapter 3 - Fig. 3.14 -  Modes of action of stem cell-based therapies in wound healing

Fig. 3.14: Modes of action of stem cell-based therapies in wound healing [127].

 

Thus, the pro-regenerative feature of stem cells makes them an attractive option in the treatment of challenging disease conditions. There is adequate proof that a stem cell-based approach can help in improving wound healing. A number of preclinical and clinical trials are being conducted that are exploring the possibility of stem cells from different sources for the promotion of wound healing and tissue regeneration (Table 3.14). These trials have demonstrated promising results and have mainly used an autologous stem cell therapy.

 

Conditions Interventions Age Phase Status
Non healing wounds Autologous BM-MSC/ fibrin spray ≥18 1 Active, not recruiting
Diabetic foot Autologous BM-MSC intramuscular/ intra-arterial injection 18–80 2 Completed; safely tolerated, wound healing observed
Second-degree burn Allogeneic BM-MSC application ≥18 1 Recruiting
Diabetic ulcer Allogeneic BM-MSC injection 18–81 1 and 2 Not yet recruiting
Critical limb ischemia Autologous CD34+ cell injection 21–80 1 and 2 Completed; safely tolerated, trends toward limb salvage with therapy observed
Critical limb ischemia Autologous BMAC injection ≥18 2 Completed; limb salvage significantly increased with therapy
Leg ulcer/gangrene Autologous peripheral blood CD34+ cell injection 20–80 1 and 2 Completed; safely tolerated, positive trends in efficacy parameters observed
Critical lower limb ischemia Autologous BM-MNC injection 18–75 1 and 2 Not yet recruiting
Chronic venous leg ulcer Autologous BMDC implantation 40–75 1 Active, not recruiting
Chronic wound Autologous ASC injection ≥18 2 Recruiting
Critical limb ischemia Autologous ASC injection ≥18 1 and 2 Recruiting
Acute burn Allogeneic hUCMSC transplantation 18–65 1 and 2 Recruiting
BMAC: Bone marrow aspiration concentrate; BM-MNC: Bone marrow-derived mononuclear cell; BMDC: Bone marrow-derived cells; BM-MSC: Bone marrow mesenchymal stem cells, hUCMSC: Human umbilical cord-derived mesenchymal stem cells.

Table 3.14: Mesenchymal stem cells in wound healing [129].

 

This section will address the various types of stem cells that have been used to heal wounds. These include mesenchymal stem cells derived from bone, adipose tissue, and hematopoietic tissue.

Bone Marrow-derived Mesenchymal Stem Cells in Wound Healing
Mesenchymal stem cells (MSCs) are the most sought after stem cells mainly due to their easy availability. MSCs are self-regenerating, multipotent stem cells with the ability to differentiate into a number of lineages such as bone, cartilage, tendon, fat, liver epithelium, lung, gastrointestinal tract, and skin cells. MSCs are also known to regulate immune response and inflammation. They are an attractive choice of cells for the treatment of numerous diseases as they possess tissue protective and reparative mechanisms. MSCs mediate their action mainly through the release of trophic factors such as vascular endothelial growth factor (VEGF), stromal cell-derived factor-1, epidermal growth factor, keratinocyte growth factor, insulin-like growth factor, and matrix metalloproteinase-9. MSCs also promote new vessel formation, recruit endogenous progenitor cells, and direct cell differentiation, proliferation, and extracellular matrix formation during wound repair [129,130].

Secretion of prostaglandin E2 by MSCs regulates fibrosis and inflammation, thereby promoting tissue healing with reduced scarring [131]. Bactericidal properties of MSCs are known to be mediated via the secretion of antimicrobial factors and by upregulating bacterial killing and phagocytosis by immune cells [132]. An overview of the mechanism of action of MSCs in wound healing is illustrated in Fig 3.14.

Exogenous MSCs have been shown to be beneficial in wound healing in a variety of animal models (Table 3.15) and have been reported to be useful in clinical cases (Table 3.14). They have also been successfully used to treat chronic wounds and stimulate stalled healing processes. Injection of BM-MSCs into excisional wounds accelerates wound closure and increases re-epithelialization, angiogenesis, and cellularity [134]. Clinical trials using alternate delivery methods have further confirmed the potential therapeutic efficacy of BM-MSCs in human cutaneous regeneration [130].

 

TICEBA - The Story of Stem Cells - Chapter 3 - Fig. 3.15 -  Mesenchymal stem cell roles in each phase of the wound-healing process

Fig. 3.15: Mesenchymal stem cell roles in each phase of the wound-healing process. Abbreviations: HGF, hepatocyte growth factor; IL, interleukin; KGF, keratinocyte growth factor; MMP, matrix metalloproteinase; PDGF, platelet-derived growth factor; TGF, transforming growth factor; TIMP, tissue inhibitor of metalloproteinases; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor [133].

 

Model Cell type Delivery method Outcomes
Deep burn wounds in rats Allogeneic and autogenic BM-MSCs Topical application New vessel and granulation tissue formation, decrease in inflammatory cell infiltration
Excisional wounds in diabetic mice Allogeneic BM-MSCs Topical application Enhanced epithelialization, granulation tissue formation, and angiogenesis
Nude rats with defected skin Human MSCs Collagen-derived skin substitute Increased wound healing
Surgical defects in mice Human BM-MSCs Fibrin polymer and intravenous Scar-free healing, little immunoreactivity
Excisional wounds in normal and diabetic mice Allogeneic BM-MSCs Intradermal injection Increased angiogenesis and cellularity
Excisional wounds in mice Allogeneic BM-MSCs Intravenous injection Accelerated wound healing
BM: bone marrow; MSCs: mesenchymal stem cells; BM-MSCs: bone marrow-derived MSCs.

Table 3.15: Animal studies on the efficacy and safety of mesenchymal stem cells in chronic wound healing [135].

 

Adipose-derived Stem Cells in Wound Healing
Adipose tissue is another potential source of adult stem cells which possess similar characteristics of BM-MSCs. Adipose-derived MSCs (ASCs) are a pluripotent, heterogeneous population of cells present in the human adipose tissue. Isolation of these cells can be easily accomplished using liposuction aspirates or excised fat samples. ASCs post purification can be differentiated into adipogenic, chondrogenic, myogenic, and osteogenic cell lineages in response to specific stimuli. Since lipoaspirates yield a very high number of ASCs, they have the additional advantage of being directly administered without in vitro expansion and differentiation. This feature makes ASCs an attractive alternative to BM-MSCs that involves complicated and painful aspiration [129].

ASCs have been tested in multiple preclinical trials on wound healing and have been found to significantly enhance cutaneous wound healing and increase blood vessel formation [136]. Autologous ASC transplantation has been proven safe and well tolerated by patients without evidence of malignant transformation over a period of 120 days [137]. A recent study by Kim WS, et al. revealed evidence indicating that adipose-derived stem cells can promote human dermal fibroblast proliferation by direct cell-to-cell contact and by secretory induced paracrine activation, which significantly accelerated the re-epithelialization of cutaneous wounds [138].

Umbilical Cord-derived Stem Cells
The umbilical cord (UC) contains two arteries and one vein, which is surrounded by mucoid connective tissue known as Wharton’s jelly. Human umbilical cord blood (UCB) is a rich source of hematopoietic stem cells and progenitor cells. Umbilical cord-derived stem cells can be extracted and purified from either the cord blood or the Wharton's jelly.

There are many advantages of UCB as a source of human stem cells (HSCs) as compared to BM and Peripheral Blood (PB). Mainly, the collection of cord blood units is easy and non-invasive for the donor and therefore, the number of potential donors is higher than for bone marrow. The umbilical cord can be easily obtained without causing pain and the procedure avoids ethical and technical issues [139].Cord blood units are stored in advance and are therefore rapidly available when needed while bone marrow has to be collected from the donor just before transplantation and there is always a risk of last minute consent refusal. Moreover, MSCs from UCB are more primitive than MSCs isolated from some other tissue sources [140].

Cord blood derived MSCs have been shown to accelerate wound healing. Umbilical cord epithelial cells also display stem cell properties and are capable of forming stratified epithelium. These cells represent a potential source of allogeneic skin graft.

Advances in Stem Cell- based Therapy in Wound Healing
Wound healing is a complex process that requires the coordinated interplay of ECM, growth factors, and cells. MSCs in particular, play an important role in mediating each phase of the wound-healing process – inflammatory, proliferative, and remodeling. Badiavas and Falanga were the first to successfully treat chronic wounds with MSC’s in 2003. The patients treated with wounds, which had previously failed to heal for more than one year, completed the wound healing process after this treatment [130].

A recent clinical trial conducted by Wettstein et al. investigated the effect of using autologous hematopoietic stem cells (HSCs) in chronic wounds in a pressure sore model [141]. This group studied three patients who received suspensions of HSC’s extracted from their own iliac crest. The treatment improved wound closure. A two-year follow-up indicated no signs of malignancy. Though the small number of patients recruited for the study was the main limitation it mainly established the relative safety of this method for chronic wound healing.

A team of scientists from Newcastle University has been able to encase adipose-derived mesenchymal stem cells in an alginate gel made from a type of brown algae, which is commonly used in food and medical applications. The greatest challenge in the practical application of stem cells in wound healing was to maintain the cells in the viable condition in the right combination of oxygen and carbon dioxide. The gel acts as a barrier from the environment and is able to sustain the stem cells for up to three days at room temperature. It can be inserted into plasters or bandages to help the faster healing of wounds like ulcers and burns. The study found that after three days at a range of temperatures (between 4 and 21°C) up to 90% of the stem cells were still viable and available for healing. This approach improves wound healing by reducing inflammation and speeding up wound closure. The findings of the study are published in the journal Stem Cells Translational Medicine [142,143].

Similar observations have also been reported by a team of researchers at the Cornell University, where they tested microencapsulated horse mesenchymal stromal cells and their potential to promote cutaneous wound healing.

Stem cells have tremendous potential to revolutionize the cosmetic industry. The use of human stem cells for cosmetic purposes is banned in Europe, China and some other parts of Asia. Most beauty brands, therefore, use plant stem cells in cosmetics. Animal-derived stem cell mediums are allowed for use in cosmetics in Singapore. CellResearch is a biotech company that has developed a range of products called Calecim that contain a combination of growth factors, cytokines, and proteins secreted red deer umbilical cord lining stem cells. According to Dr. Phan Toan-Thang, CellResearch's co-founder and associate professor of the department of surgery at the National University of Singapore's Yong Loo Lin School of Medicine, Calecim products will help boost the health of one's skin. The brand is sold in the US, Hong Kong, and Thailand [144].

CellResearch is also working on the United States Food and Drug Administration trial, using human umbilical cord lining mesenchymal stem cells to heal chronic diabetic wounds, at the University of Colorado's Anschutz Medical Center. The umbilical cord lining is a rich source of epithelial stem cells that can transform into skin tissue and mesenchymal stem cells. Umbilical cord lining stem cell treatments for severe burns and chronic bed sores are being explored in other institutions in partnership with CellResearch [144].A team headed by Prof. Martin Gasser at the University hospital in Würzburg, Germanyhas been ardently working on an interventional, single-arm, phase I/IIa clinical trial to investigate the efficacy and safety of ABCB5+ autologous, mesenchymal stem cells (MSCs) from the skin on wound healing in patients with chronic venous ulcer (CVU). The group is attempting the use of autologous MSCs isolated from a small skin biopsy and expanded in vitro, which will be applied on the wound surface of CVU under local anesthesia. The patients are being followed up for the efficacy of the treatment for three months, which allows distinguishing actual wound healing from transient wound coverage. The quality of the wound healing process will be assessed on the basis of formation of granulation tissue, epithelialization, and wound exudation. Pain will also be measured and documented on a numerical scale. To assess long-term safety an additional follow-up visit 12 months post-IMP application will also be studied [145]. This clinical trial has been supported by RHEACELL GmbH & Co. KG, which is led by the physician & the surgeon Dr. Christoph Ganss [145].

Asia is a frontrunner in the race to implement stem cell technology as a regular therapeutic approach. The multitude of clinical trials being conducted on this front will soon aid in crossing the barriers restricting the availability of stem cell therapy. Hu Ping, a cell biologist with the Shanghai Institute of Biological Science at the Chinese Academy of Sciences, was able to grow muscle stem cells to heal permanent wounds, especially those caused externally.

While animal studies and small-scale clinical studies support the potential benefit of cell therapies in wound repair, clinical barriers include a lack of sufficient clinical evidence, high costs, and a lack of standardized delivery techniques [127].

 

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News
Clinical Trials

We are now also recruiting patients for further clinical studies in phase I/IIa with allogeneic ABCB5-positive (ABCB5+) mesenchymal stem cells for the following indications: chronic venous ulcer (CVU), diabetic foot ulcer (DFU) and peripheral arterial occlusive disease (PAOD). For more information click HERE.

License

Besides the authorization to manufacture a human medicinal product in accordance with § 13 (1) of the German Medicinal Products Act (AMG) for autologous mesenchymal stem cells, TICEBA is also authorized to manufacture a medicinal product for allogeneic mesenchymal as well as allogeneic limbal ABCB5 + stem cells following a recent extension. For more information click HERE.

The Story of Stem Cells

Review our category "The Story of Stem Cells" with the newest topic "Stem cells in wound healing" HERE.

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