Stem Cells as Tools in Regenerative Therapy for Retinal Degeneration FREE

Age-related macular degeneration (AMD) affects 10% to 20% of people older than 65 years and is the leading cause of severe visual impairment in the elderly population in industrialized nations.^{1 - 2} Although several different treatment options exist for AMD, none achieves significant recovery of lost central vision. Damage to the retinal pigment epithelium (RPE) resulting in atrophy is a critical feature of AMD. Alterations in the RPE monolayer are part of physiological aging, as well as pathophysiologic processes. There are several characteristics of a normal but aged pigment epithelium and these include a decrease in RPE density, a clinically observed decrease in the pigmented appearance of the RPE cells, and the accumulation of lipofuscin within RPE cells.³ In AMD, the initial morphologic changes are associated with the formation of drusen and other deposits on Bruch's membrane. Subsequently, RPE cell loss occurs presumably via apoptosis associated with the loss of cell attachment.⁴

Data suggest that widespread oxidative damage occurs in the retina of patients with advanced geographic atrophy.⁵ Proteins and nutrient metabolites related to oxidative stress are also upregulated in retinas of patients with AMD.^{6 - 7} Therefore, oxidative damage is thought to play a major role in advancement of RPE loss, either as a pathogenic factor or as evidence for a major deficiency in the oxidative defense system. Additional reports support a role for the immune system, including complement activation, whereby accumulation of extracellular plaques and deposits elicits a local chronic inflammatory response that exacerbates the effects of primary pathogenic stimuli, in the development of drusen and the pathogenesis of AMD.^{8 - 10} Areas of bare Bruch's membrane are also found after submacular surgery for choroidal neovascularization performed in exudative or “wet” AMD.^{11 - 14} Furthermore, damage to the RPE has been also observed after uncomplicated photodynamic therapy, an established treatment for choroidal neovascularization.¹⁵ Degenerative changes within the neurosensory retina and/or choriocapillaris occur simultaneously to the RPE atrophy.¹⁶ Fundus changes may predispose the eye to develop the neovascular/exudative stages of AMD and lead to irreversible severe vision loss.⁹ Although treatment options exist for wet AMD, eg, injection of ranibizumab providing visual improvement in 25% to 40% of patients with choroidal neovascularization,^{17 - 18} none for dry AMD has been adapted.¹⁹ Thus, transplantation of RPE cells to fill in this defect before irreversible atrophy of the foveal photoreceptors occurs has been pursued. However, it has been shown that allogeneic RPE cells do not attach to senescent Bruch's membrane efficiently and do not undergo proliferation and spreading to fill in the defect.^{20 - 21} Even under circumstances in which allogeneic RPEs do attach and proliferate on aged Bruch's membrane, their ability to survive long-term is compromised.²² Transplantation of autologous RPE cells has also been explored in man, but evidence of functional recovery has not been achieved.²³

Hereditary retinal degenerations, such as retinitis pigmentosa, are major causes of blindness in the Western world, with an incidence of 1 in 2000 individuals.²⁴ The progressive loss of vision is due to mutations in more than 100 identified genes and affects different cellular compartments in either the photoreceptor cells (PRCs) themselves or the underlying RPE.²⁵ The disease is characterized by the onset of night blindness, an early loss of the peripheral visual field, and the late loss of central vision.²⁶ Different therapeutic strategies have been developed, and in recent years, major advances were reported to rescue retinal degeneration in animal models, such as mutation-specific gene-based treatments (gene replacement/gene silencing),³ mutation-independent approaches such as the administration of neurotrophic factors (genetically expressed and subretinally injected), or administration of donor cells (PRCs, RPE cells, stem cells [SCs]).^{27 - 29} At the same time, the search for a mutation-independent pharmacological treatment has been intensified, especially after reports of the neuroprotective potential of various growth factors (eg, glial-derived neurotrophic factor or ciliary neurotrophic factor) emerged.^{30 - 31} The latter, a phase 1 trial in patients with retinitis pigmentosa, indicated that ciliary neurotrophic factor is safe for the human retina, even with severely compromised photoreceptors, and showed visual improvement in the study.

Regenerative medicine seeks new therapies for patients with severe injuries or chronic diseases, including congestive heart failure, osteoporosis, Alzheimer and Parkinson diseases, spinal cord injuries, age-related macular degeneration, and retinitis pigmentosa. More than 100 million Americans have such illnesses and many of them have few or no treatment options.³² In all of these disorders, the body's own repair mechanisms are not able to restore diminished function. Therefore, the main focus of regenerative medicine is the development and manufacturing of tissue and/or organ replacements to avert pathological detriment and allow restoration of physiological function. Several approaches for promoting regeneration have been pursued to date: promotion of endogenous regeneration via therapeutic use of growth factors, exogenous delivery of living cells of both allogeneic and autologous origin, tissue engineering, and the development of artificial organs.

This review will focus mainly on the use of living cells for regenerative medicine, their sources in general, and the challenges associated with their use. Stem cells are of interest because of their plasticity and the capacity to self-renew as well as to give rise to specialized cell types. They remain uncommitted and self-renewable until they receive a signal(s) to develop into distinct cell types.³³ In addition, because SCs can proliferate indefinitely in their undifferentiated state, they are expected to alleviate the problem of the shortage of donor cells for cell replacement therapy.³⁴ The current challenges in SC-mediated regenerative therapy are how to maintain the stemness of the cells while promoting regeneration, identifying the optimal source of cells, controlling host vs donor alloreactivity if allogeneic cells are used, and promoting efficient function while preventing loss of control of regulation that could result in teratoma formation.³⁵

A different approach to replace degenerated cells would be the use of reprogrammed somatic cells. For this, several strategies, such as nuclear transplantation, cellular fusion, introduction of defined transcription factors, and culture-induced reprogramming have been used.³⁶ Thereby, conversion of differentiated cells into an embryonic state with an increase in potency has been induced. Nuclear reprogramming has special therapeutic potential as it can be used to create patient-specific cells.

Furthermore, combining SCs and progenitor cells with bioengineering to generate tissue equivalents in culture is another promising therapeutic strategy. Replacement of tissue lost to disease or trauma using transplants delivered on polymer scaffolds can be applied to improve survival and promote the organized differentiation of grafted cells. This might be especially important in models of retinal degeneration and injury to create the appropriate architecture for integration of transplanted retinal SCs and precursor cells. Results have shown that the scaffold may promote differentiation of retinal precursor cells and is associated with increased cell survival.³⁷

In 1998, human cells with the pluripotent property of embryonic SCs (ESCs) were isolated.^{38 - 39} In general, ESCs show the following characteristics: they (1) can be isolated from the inner cell mass of the blastocyst; (2) proliferate extensively in vitro; (3) maintain a normal euploid karyotype over extended culture; 4) differentiate into derivatives of all 3 germ layers; 5) express high levels of Oct4; and 6) show telomerase activity.⁴⁰ Embryonic SCs are believed to hold great promise for clinical applications for the replacement of diseased or degenerating cell populations, tissues, and organs. Numerous differentiated cell types have been derived from ESCs, including neural tissue,^{41 - 42} endothelial cells,⁴³ blood cells,⁴⁴ cardiomyocytes,⁴⁵ and hepatocytes.⁴⁶ In addition, different ESC lines have been established and several methods for culture, maintenance, and the generation of different cell types of all germ layers have been developed.⁴⁰ In regard to ocular tissue, however, reproducible and efficient methods to engineer either a monolayer of RPE cells or PRCs within an integrated neural network are still lacking. In general, many issues remain to be solved before ESCs can be harnessed as therapeutic regenerative tools. Undifferentiated ESCs in a graft have the potential to form teratocarcinomas in the recipient organism after activation to differentiate. The optimal signals to control differentiation need to be defined.⁴⁷ Furthermore, there is still the controversy in some venues over the ethical justification for using ESCs in research.⁴⁸

Embryonic SCs are not the only SC candidates for generation of differentiated cell types.⁴⁹ Other pluripotent cell types include SCs derived from the primordial germ cells of the gonadal ridge⁵⁰ and possibly cancer SCs, which recently have been identified in leukemia and several solid tumors.⁵¹ In the past decade, researchers have also defined committed SCs or progenitor cells from various tissues in both adult animals and humans.⁵² The so-called adult SC can give rise to cells of a particular tissue and include, for example, bone marrow (BM)–derived SCs (BMSCs), one of the earliest clinically used and most widely studied SC populations.⁵³ These multipotent SCs differentiate into cell types from the germ layer of their origin. However, it has been found that they are generally less effective than ESCs in generating other lineages (Figure 1A).

Today, it is widely agreed that several subsets of SCs can be found in the BM: (1) hematopoietic SCs, the source of all blood cells; (2) mesenchymal SCs, nonhematopoietic stromal cells that differentiate into mesenchymal tissues; and (3) tissue-committed SCs (TCSCs) that are already committed to neural, cardiac, and other lineages.⁵⁴ Figure 1B demonstrates the population of CD45⁻ TCSCs. Recent publications showing the existence of CD45⁻ TCSCs have led to renewed interest in adult BMSCs as a source for tissue repair in vivo.⁵⁵

Pluripotency and differentiation potential of BMSCs, even across germ layers, have been extensively examined in vitro and in vivo.^{56 - 60} In the central nervous system, multipotential precursors for neurons, astrocytes, and oligodendrocytes have been found. These so-called neural SCs from both the fetal and adult central nervous system have proliferated in vitro and can differentiate to show morphological and electrophysiological features characteristic of neural cell types.^{61 - 62} For cell replacement therapy in the ocular system, the isolation and characterization of retinal SCs (RSCs) in 2000 represented an important step forward. Tropepe and colleagues⁶³ reported the identification of a SC in the adult mouse eye that represents a possible substrate for retinal regeneration. Retinal SCs are localized in the pigmented ciliary margin and clonally proliferate in vitro to form spherical colonies of cells that can differentiate into retinal-specific cell types, including rod photoreceptors, bipolar neurons, and Müller glia. Other organs seem to also contain cell types with SC-like properties, including the heart⁶⁴ and islet.⁶⁵ Taken together, these results provide evidence that SCs exist within the adult mammalian organism and that there is an endogenous program to incorporate these cells into damaged structures. Although effective at maintaining existing tissues and organs, it appears that this endogenous physiological mechanism is not capable of repairing significant damage.

Tissue replacement in the body takes place physiologically by 2 mechanisms. One is the replacement of differentiated cells by newly generated populations derived from residual cycling SCs. Blood cells are a typical example of this kind of regeneration. Whole hematopoietic lineage cells are derived from a few self-renewing SCs by regulated differentiation under the influence of appropriate cytokines and/or growth factors.⁶⁶ The second mechanism is the self-repair of differentiated functioning cells preserving their proliferative activity. Hepatocytes, endothelial cells, smooth muscle cells, keratinocytes, and fibroblasts are considered to possess this ability. Following physiological stimulation or injury, factors secreted from surrounding tissues stimulate cell replication and replacement. In contrast, those cells that are more fully differentiated are limited in terms of their proliferative potential by senescence and by their inability to incorporate into remote target sites.⁵² On the other hand, differentiation of SCs, ESCs as well as adult SCs, and transdifferentiation of local terminally differentiated cells have been shown under many circumstances in vitro and in vivo.⁶⁷ For regenerative medicine to be successful, the key point is to combine physiological properties of SCs with directing the differentiation into the desired cell types via environmental cues.

The mechanisms for SC-mediated differentiation events, including documented functional recovery, are still under considerable scientific debate. For adult SCs, the controversy between transdifferentiation and fusion has still to be solved. Recently, it was reported that BMSCs are able to “transdifferentiate” or change commitment into cells that express early heart, skeletal muscle, neural, or liver cell markers.^{56 - 59 ,64} Similarly, SCs from the BM contributed to the regeneration of infarcted myocardium.^{68 - 69} This was supported by the observations in humans that transplantation of SCs from mobilized peripheral blood expressing the early hematopoietic CD34⁺ antigen led to the appearance of donor-derived hepatocytes, epithelial cells, and neurons.⁷⁰ Therefore, it was initially presumed the repair seen in damaged host tissues following SC transplantation or homing was due to incorporation and transdifferentiation of the BMSCs at the sites of damage. However, a number of studies have challenged this concept, providing evidence that BMSCs may instead incorporate into host tissues via fusion with host cells.^{71 - 72} One of the key studies demonstrating evidence for fusion was that of Alvarez-Dolado et al,⁷³ where they examined CD45⁺ BMSCs and found that essentially all integration of these cells into host tissues involved fusion. Because the CD45⁺ subset of BM-derived cells is now thought to be committed to the hematopoietic lineage (as opposed to CD45⁻ cells, which give rise to other nonhematopoietic lineages), one could argue that Alvarez-Dolado et al did not examine the appropriate subset of cells capable of contributing to nonhematopoietic tissues. Additionally, they only examined incorporation of CD45⁺ cells into normal tissues, as opposed to sites of damage, where the mechanism of integration may be different because of the inflammatory milieu. Nevertheless, other studies have also found evidence that SCs can fuse with host cells, forming heterokaryons.⁷¹ Such studies raise the question as to whether the transdifferentiation of BMSCs seen in culture really occurs when these cells are injected in vivo or whether their primary therapeutic contribution is instead via fusion with host cells. Other reports found no proof of host cell fusion when SCs differentiate in the host.⁷⁴ Further, there is evidence that a single BMSC can repopulate the BM, giving rise to all hematopoietic precursors, and that such cells could in turn contribute to other nonhematopoietic tissues. The question of fusion with host cells vs transdifferentiation in vivo is the topic of several comprehensive reviews and reports that conclude that definitive experiments on this topic are lacking.^{75 - 76} Therefore, it remains unresolved as to whether BMSCs integrating into damaged tissues undergo transdifferentiation in vivo to express markers of differentiation or whether they express such markers as a result of fusion with endogenous host cells.

Stem cell–based therapy has been tested in animal models for several diseases, including neurodegenerative disorders, such as Parkinson disease, spinal cord injury, and multiple sclerosis.^{77 - 79} The replacement of lost neurons that are not physiologically replaced is pivotal for therapeutic success. In the eye, degeneration of neural cells in the retina is a hallmark of such widespread ocular diseases as AMD and retinitis pigmentosa. In these cases, the loss of photoreceptors is the primary cause of blindness. This can result from dysfunction in either the PRCs or the underlying RPE that supports their survival.

Transplantation of RSCs with the potential to generate new retinal cells provides an alternative approach to enable the replacement of lost PRCs or RPE. Retinal SCs may restore vision in patients who have degenerative retinal diseases by 2 possible means: (1) repopulation of the damaged retina (eg, PRCs) and/or (2) rescue of retinal neurons from further degeneration.⁸⁰ Different research groups have successfully isolated murine putative RSCs from the ciliary margin and human RSCs in the pars plana and pars plicata.^{81 - 82} However, the transplantation of these cells in normal and degenerative rodent retina was only minimally successful because of the limited ability of the cells to invade and integrate into the host retina.²⁷ On the other hand, transplantation of immature postmitotic rod precursors from the developing retina (postnatal day 1) improves retinal integration.⁸³ MacLaren et al⁸³ showed that timely selection of the progenitors is pivotal for success. The optimal result occurs when selected cells were biochemically committed but not yet morphologically differentiated. The capability of subretinally or intravitreously injected RSCs to invade and integrate into the neural retina remains restricted to sites of retinal injury. Breakdown of physical barriers, such as the outer limiting membrane, and/or release of unknown neurotrophic factors are most likely required to stimulate RSC integration.⁸⁴ To date, only sparse data are available regarding factors that might stimulate migration, integration, and differentiation of RSCs into the neural retina. However, it is assumed that neurotrophic factors, such as transforming growth factor,⁸⁵ fibroblast growth factor,⁸⁶ or epidermal growth factor,^{87 - 88} might play a role. Recent evidence has suggested that hepatocyte growth factor/scatter factor, a pleiotrophic factor with mitogenic and morphogenic activities, may also be involved in the development and maintenance of neurons and PRCs.⁸⁹

In AMD, the replacement of diseased RPE would be pivotal to protect or rescue the adjacent PRCs. Unfortunately, no convincing animal model for AMD exists to date. Therefore, the sodium iodate (NaIO₃) model of RPE damage, established by Korte et al⁹⁰ in 1984, has been used to study at least the repopulation of bare areas of normal Bruch's membrane.⁹¹ Briefly, the selective and patchy degeneration of the RPE monolayer after intravenous NaIO₃ injection is directly correlated with decreased visual function, decreased electrophysiological function, and anatomical cell loss in the RPE (and subsequently in the retina) (Figure 2). Furthermore, the extent of the RPE damage is time and concentration dependent, as we recently published.⁹² Interestingly, NaIO₃-damaged RPE cells express higher amounts of cytokine/growth factors involved in SC homing. After treatment with NaIO₃, murine RPE cells express higher levels of stromal-derived factor 1 (SDF-1), as well as other signaling factors (complement factor C3 and hepatocyte growth factor/scatter factor). Stromal-derived factor 1 is a chemokine whose receptor CXCR4 is expressed on BM-derived progenitor cells and SCs.⁹³ While there was no evident change in expression of vascular endothelial growth factor and RANTES (regulated on activation, normal T-cell expressed and secreted), there was increased expression of the cytokine leukocyte inhibitory factor, known to promote self-renewal in ESCs.⁹⁴ Furthermore, supernatants of NaIO₃-damaged RPE exert a priming effect on BMSC migration in vitro as they enhance their transwell migration.⁹⁴ These results provide evidence that damage to the RPE leads to production of soluble factors that can cause specific chemotaxis of BMSCs and raises the possibility of their recruitment to the site of damage. These data support the possibility of using BMSCs to replace damaged cells, especially RPE, in eyes with retinal degenerations. To investigate this further, we have undertaken endogenous as well as exogenous approaches using BMSCs using the earlier-described NaIO₃ model. Endogenous refers to existing BM cells in the host while exogenous refers to adoptively transferred cells.

Two types of approaches can be used to promote SC-mediated regenerative repair of RPE: endogenous and exogenous. Endogenously, RPE injury combined with pharmacologically enhanced growth factor–mediated mobilization lead to migration of BM-derived cells into the subretinal space. Bone marrow–derived SCs (c-kit⁺), macrophages (F4/80), and leukocytes, such as granulocytes and monocytes (CD11b), could be identified. Thereby, the number of c-kit⁺ BMSCs in the eye after NaIO₃ injection and mobilization increased dramatically compared with the mobilized control mice who did not have RPE damage.⁹¹ The migrated BMSCs had incorporated in a monolayer along the RPE 4 weeks after transplantation and expressed the RPE markers RPE65 and microphthalmia-associated transcription factor (MITF) (Figure 3). These findings suggest that BMSCs are attracted to damaged RPE and are induced to differentiate into components of RPE. Mobilization enhances the outcome.

These results demonstrated that a physiological process is in place in vivo to recruit SCs to the damaged RPE and that endogenous BM-derived cells are able to integrate into the damaged RPE and express markers of RPE differentiation. Nevertheless, the significant experimental damage to the RPE could not be repaired by this endogenous approach, nor does this endogenous program appear capable of repairing or preventing the progressive damage to the RPE that occurs in AMD and retinitis pigmentosa. Thus, it appears that such recruitment of endogenous cells may not be sufficient to physiologically repair significant damage to the RPE in the same fashion that recruitment of endogenous SCs cannot repair major damage to the spinal cord or heart.

To optimize the number and availability of circulating BMSCs, we then examined an exogenous approach for regeneration of damaged RPE. Additionally, this allowed us to define the precise cell types involved, using cell sorting as opposed to the mixture of SCs and other BM-derived cells mobilized into the periphery with the endogenous approach. We injected fluorescent-activated cell–sorted BMSCs with the phenotype lin⁻ (negative for all lineages of differentiated BM cells), SC antigen 1 (Sca-1)⁺ intravenously into NaIO₃−treated animals. The BMSCs could be detected in the subretinal space on Bruch's membrane in areas of RPE loss on day 4 after cell injection, whereas controls without NaIO₃ injection showed no BMSCs. The double staining for Sca-1 and green fluorescence protein confirms the BM origin of the cells systemically transferred and confirms that hematopoietic SCs home to the area of damaged RPE after NaIO₃ injection. One and 2 weeks after transfer, BMSCs could be identified in the subretinal space but they did not express RPE markers. Immunocytochemical staining showed the expression of RPE65 in BMSCs 4 and 6 weeks after transplantation. These results suggest that, as with the endogenous cells, BMSCs injected systemically into the host home to the site of damage, where they integrate and express markers of RPE differentiation in a time-dependent fashion.⁹⁵ One critical area of research is to define the optimum milieu in which to promote the potential for repair.

A third route for BMSC delivery is by direct subretinal injection. We found that subretinally injected BMSCs integrated into the RPE and expressed markers of differentiation (eg, RPE65). The optimal route for SC delivery remains to be determined. Concentrating the cells might provide a kinetic advantage for incorporation of the cells into the altered tissue. Thereby, the cells would not have to home to sites of damage from the circulation.

However, the use of SCs to replace degenerated RPE cells has not yet demonstrated the ability to rescue PRCs at risk of damage. If SC differentiation and reconstitution of the damaged RPE monolayer occur after photoreceptor degeneration, a rescue effect will not be possible. Alternatively, if the mobilization of endogenous SCs occurs continuously or over a prolonged period, photoreceptor damage and/or rescue may be possible.⁹⁶

The regenerative capability of BMSCs in the ocular system is not only restricted to RPE replacement. Chiou et al⁹⁷ showed that BMSCs have multilineage differentiation potential in vitro and differentiate into retinal cells and photoreceptor lineages after coculture with RPE cells. Other groups have followed different approaches to replace diseased RPE cells. Haruta and colleagues⁹⁸ harvested RPE-like ESCs in vitro and achieved functional improvement after subretinal transplantation into Royal College of Surgeons rats.

Because only a small percentage of total BM cells are chemoattracted to supernatants from damaged RPE in vitro, as well as into damaged RPE in vivo, the properties of this subset of BM-derived cells need to be considered. Recent data indicate that the CD45⁺ population of SCs is committed to hematopoietic lineages, while the CD45⁻ population is believed to remain pluripotent and thus capable of differentiation into various nonhematopoietic tissues.⁹⁹ Kucia et al⁵⁵ showed that CD45⁻ BMSCs are composed of subsets of cells already committed to skeletal muscle, heart muscle, liver, and neural tissues. These so-called TCSCs, more recently renamed very small embryonic-like (VSEL) cells,¹⁰⁰ express Oct4, an SC marker, in addition to markers of tissue-specific progenitors. These TCSCs are mobilized into peripheral blood during organ injury.¹⁰¹ Stromal-derived factor 1–based chemotactic isolation combined with real-time reverse transcriptase–polymerase chain reaction analysis of messenger RNA revealed that early TCSCs (1) reside in the normal human and murine BM; (2) express CXCR4 on their surface; and (3) can be highly enriched in humans and mice after chemotaxis to a SDF-1 gradient. These studies were performed on freshly isolated cells, ruling out the potential contribution of culture-related transdifferentiated hematopoietic SCs or mesenchymal cells. In our experiments, we found that Sca-1⁺ CD45⁻ BMSCs are highly enriched in messenger RNA for retinal/RPE progenitors (Six-3, OTX, Pax-6, MITF; data not shown) and, furthermore, that this is the subset of BMSCs that has migrated in response to supernatants from damaged RPE in transwell assays. Thus, it appears that RPE-committed VSEL cells (approximately 0.05% of the population) are present within the Sca-1⁺ CD45⁻ subset of BMSCs. This is supported by data from in vitro experiments using a coculture of BMSCs and RPE cells to trigger SC differentiation into the RPE lineage (Figure 4).⁹¹ The BMSCs changed their morphology from round to epithelial-like and expressed the epithelial markers cytokeratin; MITF, expressed on common progenitors of retina and RPE and persisting expression following RPE differentiation (its expression diminishes in cells that progress along a retina lineage); and the RPE-specific marker RPE65 after 2 weeks (Figure 5). The process required direct cell-cell contact between BMSCs and RPE. No staining for RPE markers was detected when a membrane separated both populations of cells. This was a specific effect, as no positive staining was detected when RPE cells were replaced with fibroblasts.⁹¹

Finally, degenerations in the mammalian retina, initiated by defects in photoreceptors or RPE, often leave the neural retina deafferented. It responds to this challenge by remodeling, first by subtle changes in neuronal structure and later by large-scale reorganization, and represents the invocation of mechanisms resembling developmental and central nervous system plasticity. This neuronal remodeling and the formation of a glial seal may abrogate many cellular and bionic rescue strategies. On the other hand, survivor neurons appear to be stable, healthy, active cells, and given the evidence of their reactivity to deafferentation, it may be possible to influence their emergent rewiring and migration habits.¹⁰²

While it has been convincingly demonstrated that various embryonic and adult mouse and human SCs have differentiation capabilities, how to harness the full regenerative potential remains to be determined. Characterization of SC phenotypes is more complicated as just the assessment of specific markers. Additionally, immunological and tumorgenic concerns hamper the therapeutic use of ESCs. Adult SCs are free from the ethical concerns but are usually available in very limited numbers and their numbers decrease with age. Furthermore, their differentiation potential is still hotly debated. In vitro modulation of SCs might show possible solutions to overcome these obstacles. Thereby, one could maintain the cells undifferentiated for a prolonged time, expand cell numbers, and promote differentiation along a desired lineage to suppress tumorigenicity or facilitate “transdifferentiation.” The culture milieu provides the means to control the microenvironment as well as to specifically alter it. Thereby, soluble factors, collaborative cells, and biomaterials are currently widely used as environmental cues to affect SC fate.¹⁰³ Alternatively, growth factor (granulocyte colony-stimulating factor)^{104 - 105} and/or cytokine (Flt3L)¹⁰⁶ mobilization of endogenous SCs, BM derived or tissue specific, and their subsequent homing along a SDF-1¹⁰⁷ and/or complement C3⁶⁶ gradient to the site of damage could be used. However, many kinetic and quantitative issues have yet to be solved.

Currently, a number of experimental SC treatments are being evaluated, including research in cancer, Parkinson disease, cardiac disease, Huntington disease, multiple sclerosis, and acute spinal cord injury.^{108 - 109} Although a number of challenges must still be addressed, the potential impact of SC-based regenerative medicine holds great promise.

Correspondence: Suzanne T. Ildstad, MD, Institute for Cellular Therapeutics, University of Louisville, 570 S Preston St, Ste 404, Louisville, KY 40202-1760 (stilds01@louisville.edu).

Submitted for Publication: November 3, 2008; final revision received January 26, 2009; accepted February 3, 2009.

Financial Disclosure: None reported.

Funding/Support: This work was supported in part by National Institutes of Health grants R01 DK069766 and 5RO1 HL063442; Juvenile Diabetes Research Foundation grants 1-2005-1037 and 1-2006-1466; The Department of the Navy, Office of Naval Research; The Department of the Army, Office of Army Research (this publication was made possible by award W81XWH-07-1-0185 from the Office of Army Research); the National Foundation to Support Cell Transplant Research; the Commonwealth of Kentucky Research Challenge Trust Fund; the W. M. Keck Foundation; The Jewish Hospital Foundation; and Research to Prevent Blindness.

Disclaimer: Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the Office of Army Research.

Additional Contributions: Luisa M. Franco, MD, and Yang Li, MD, provided technical assistance, and Carolyn DeLautre prepared the manuscript. We thank the staff of the animal facility for outstanding animal care.