The predominant paradigm for the use of stem cells in bone disorders has been centered on the strategies for reconstructing segmental regions of the skeleton, lost to trauma or surgery, and in muscle diseases, to provide a normal population of cells to replace defective tissue. Although this tissue-engineering approach meets a huge clinical need as well as a potentially large market, one of the most seductive promises arising from the notion that musculoskeletal tissues emanate from a system of postnatal progenitors resides in the hope of providing a cure for severe, crippling genetic diseases. Tackling these diseases involves cell therapy and gene therapy strategies rather than tissue engineering. Although the target diseases in this area are usually rare, their social and human cost is huge. Whereas the targeted diseases may be rare themselves, their underlying mechanisms often include derangements in ubiquitous pleiotropic pathways. Therefore, wide windows for pharmacological intervention in several settings may be opened as a fallout of such studies.

There are three important ways in which postnatal stem cell research can contribute to the future cure of crippling diseases of the skeleton and of skeletal muscle. First, current progress in the definition of the anatomical identity, ontogeny, and phenotype of postnatal progenitors in these tissues provides the long-missing, fundamental awareness of which cell type and of how one wants to harvest, purify, grow, or deliver in view of a cell therapy approach. As we emerge from the “mesenchymal stem cell” paradigm, we realize through current experimental data that a system of tissue-specific, self-renewing progenitors exist, with a similar anatomy and surface phenotype, in the microvascular system of different tissues. In bone and skeletal muscle, the isolation of MCAM-expressing clonogenic subendothelial cells coincides with the isolation of enriched populations of skeletogenic and myogenic progenitors, respectively. Awareness of the nature of postnatal progenitors also provides a novel angle on their therapeutic use—to be centered on the mimicry of their in vivo physiological function in addition to their regenerative potential. 

Second, stem cells provide model systems in which to seek the downstream effects of known mutations, which mediate the development of a disease phenotype. This process provides important clues for the subsequent design of novel types of drugs in ways that would even appear counterintuitive based on the mere awareness of the identity of the causative mutations. For example, the genomics of human transgenic stem cells created by permanent lentiviral transduction, with the causative gene of fibrous dysplasia (FD, OMIM#174800), has revealed multiple and at times unsuspected determinants of the development of organ lesions.

Third, stem cells provide the natural target for the correction of gene defects in self-renewing tissues. Tools are being developed that will allow us to tackle even the most challenging scenarios in gene therapy, such as the selective silencing in stem cells of dominant, gain-of-function mutations in ubiquitously expressed genes, such as the FD-causing gene. More than the direct translation of preclinical studies to gene therapy approaches in the clinic, it is the insight into the mechanisms and requirements that arise from “in-stem cell” experiments that represents the most important added value of this approach at the current time.

Pluripotent stem cells isolated from embryos (ES cells) or by the direct reprogramming of somatic cells (iPS cells) represent an inexhaustible source of precursor cells that can be differentiated into specific cell lineages. As with conventional organ transplants, cell-based therapies will face immunologic barriers. Genetically matched pluripotent embryonic stem cells generated via nuclear transfer (ntES cells) or parthenogenesis (pES cells) are a possible source of histocompatible cells and tissues. In a proof of principle experiment, we have shown that customized ntES cells can be used to repair a genetic immunodeficiency disorder in mice (Rideout et al., Cell, 2002). However, the generation of ES cells by nuclear transfer remains inefficient and to date has not been achieved with human cells. ES cells with defined histocompatibility loci can be generated at much higher efficiency by the direct parthenogenetic activation of the unfertilized oocyte (Kim et al., Science, 2007). Subsequently, cell lines can be genotyped and selected for major histocompatibility complex (MHC) identity to the oocyte donor. Cell lines with homozygous MHC haplotypes can also be identified, and tissues from such cells engraft in MHC heterozygous recipients. Compared with ES cell lines from fertilized embryos, pES cells display comparable in vitro hematopoietic activity, and blood derivatives can repopulate hematopoiesis in irradiated adult mouse recipients. These experiments establish murine models for generating histocompatible ES cell-derived tissue products and suggest the theoretical feasibility of ES cell banking to enable off-the-shelf cell therapies. We have generated human iPS cells by the direct reprogramming of human somatic cells, with retroviruses carrying OCT4, SOX2, MYC, and KLF4 (Park et al., Nature, 2008). Although this represents a platform for generating customized, patient-specific cells for research, nonviral methods must be established for making human iPS cells before such cells are available for human clinical applications. Strategies for generating patient-specific iPS cells and for their use in research and therapy will be discussed.

The treatment of multiple sclerosis (MS) has evolved over the past decade from temporizing measures involving getting over individual disease relapses to a more aggressive approach to stem the early inflammatory events that are thought to contribute to the ultimate demise of the neurons and axons that underlies the inexorable progression seen by most patients. However, even these anti-inflammatory measures, which have nurtured the development of more disease- and immune-specific therapies, have failed to stem the transition to progressive disease, probably because the cumulative injury inflicted to the central nervous system (CNS) is inadequately repaired. These results have led researchers to begin disease-specific therapy as early as possible, but recent evidence shows that starting treatment even after the first presentation of “possible” MS does not prevent the development of disease progression. 

The recipe for the successful treatment for MS probably entails curtailing the development and recurrence of inflammatory CNS attacks by the immune system but at the same time promoting the protection, repair, and ultimate regeneration of the damaged CNS elements. This has led researchers to look at the possibility of combining a therapeutic approach that will both halt the autoimmune attack on the CNS (i.e., immunosuppression) and provide a source of potential repair through the introduction of stem cells. In fact, the use of bone marrow-derived stem cells was an essential component of aggressive immunosuppression using some of the current day chemoablative regimens, providing the necessary “rescue” treatment, without which continued viability was at risk due to a complete wipeout of the hematopoietic system. These regimens, in various forms, have been used for the past decade in the treatment of MS with varying results. More recent attempts, however, may be revealing that by completely staving off further CNS inflammatory attacks and by providing a healthy environment for the regrowth of the immune system, unfettered by continued disease-specific immunotherapy, there is a chance for CNS repair or regeneration in MS.

A spectrum of engineering approaches has facilitated deployment of cell-based therapies for metabolic organs over the past several decades. At one end of the spectrum are organ or tissue replacement strategies that involve membrane-based devices seeded with cells from human or animal donors. For pancreatic islet replacement, a vigorously pursued strategy has been to encapsulate donor islets in semi-permeable membranes that allow diffusion of insulin and small molecules, but inhibit immune destruction of the cells. This approach has illuminated facets of the response to implanted islets but has not yet been successfully clinically translated. For liver replacement, adaptation of dialysis-type reactors to incorporate liver cells has reached an advanced stage and progressed to clinical trials for treatment of liver failure, but these technologies remain commercially challenging to develop. In parallel to traditional device-based approaches, advances in cell biology have spurred engineers to develop quantitative engineering models of how cells respond to cues in their environment, laying the foundation for designing cellular microenvironments that can direct cell survival, growth, and differentiation. These types of engineering models are beginning to yield insights into how to manipulate cell behavior to improve cell survival and integration into tissues following transplant in vivo, thus enabling regenerative rather than replacement technologies to be developed. Engineering models also provide the blueprints for design of biomaterials and scaffolds that can best facilitate tissue and organ regeneration. Finally, with the advent of new tools in microfabrication and microfluidics, there is rapidly growing emphasis on building in vitro models that capture the complexity of in vivo cell–cell interactions in 2–D and 3–D, allowing investigation and manipulation of human cells and tissues in ways that provide insight into physiology and pathophysiology. Development of accesible in vitro models of human organs may facilitate development of therapies that prevent progression of chronic diseases that affect metabolic organs, or lead to cures that reduce the need for organ replacement.

Cell replacement therapy is an attractive approach for several liver diseases currently treated by whole-organ transplantation. Each hepatic disorder has its own therapeutic threshold, ranging from low levels of hepatocyte replacement in clotting disorders to a requirement of >50 percent for urea cycle disorders. Clinical trials of hepatocyte transplantation have been carried out and have generally been found to be safe but subtherapeutic. The challenge for clinical success lies in enhancing the degree of donor cell contribution, which will require strategies for host liver conditioning that enable high initial survival, the engraftment of transplanted cells, and the selective growth of the donors. Similarly, liver cirrhosis is a feature of most liver diseases leading to transplantation. Cell therapy will have to be combined with strategies to eliminate fibrosis and scarring if it is to be successful in those conditions.

Glial-restricted precursors (GRPs) are lineage-restricted precursors of the central nervous system (CNS) glial cells and have the potential to differentiate into oligodendrocytes and astrocytes. In rodent demyelinating and injury models, GRP-derived oligodendrocytes remyelinate demyelinated axons, and GRP-derived astrocytes secrete growth factors that stimulate protection and axonal sprouting of damaged axons. We believe the inherent characteristics of GRPs render them a natural means to repair defects in myelin production in the CNS and thus may be an ideal therapeutic for CNS diseases, such as transverse myelitis (TM) and multiple sclerosis (MS). 

However, we and other groups have found that when transplanted into an inflammatory demyelinated lesion (rather than one from chemical or genetic causes), GRPs do not fully differentiate and myelinate nearly as well as when transplanted into other types of demyelinated lesions. When GRPs encounter the inflammatory environment seen in TM and MS patients, the differentiation of these cells halts, and they fail to repair myelin. I will review the preclinical data using GRPs in inflammatory models of demyelination. 

In a separate series of studies, we are transplanting rodent and human embryonic stem (ES) cell-derived motor neuron progenitors into various models of motor neuron disease. The basis for this project is our previous findings that the transplantation of mouse ES cell-derived motor neurons into paralyzed rats results in the connection of transplant-derived motor axons with host skeletal muscle and in the functional return of movement. We have developed a focal model of weakness in dogs using the motor neuron-specific lectin ricin and will transplant human ES cell-derived motor neuron progenitors into the spinal cord of weak dogs. Selected animals will receive intraspinal growth factors to increase the efficiency of synapse formation onto the transplanted cells and other drugs to enhance the ability of transplanted axons to reach target muscle. Some animals will receive additional growth factors in skeletal muscle to further attract transplant-derived axons to reach and form functional connections.

Ultimately, this study will offer insight into the biology of demyelination and paralysis and will identify potential therapeutic strategies for these disorders.

As the population ages and the acute mortality from cardiovascular disease decreases, a large population of patients is emerging who have symptomatic chronic ischemic vascular disease, many of whom remain severely symptomatic despite exhausting conventional medical therapy and mechanical revascularization. In addition, mounting evidence suggests that microvascular insufficiency plays a significant role in the pathophysiology of ischemia. At the present time, there are no therapies that directly address the needs of this patient population.

Pre-clinical and early clinical data indicate that a variety of growth factors and stem/progenitor cells may be employed therapeutically for repair of ischemic tissue.

Preclinical studies documented the potential therapeutic potency of endothelial progenitor cells, both as cultured and freshly isolated cells. Early phase clinical trials using a variety of approaches have been completed providing data of feasibility, safety and bioactivity. Later phase trials are under way.

Accordingly, the goal of ischemic tissue repair appears feasible and is being approached in human clinical trials. The evolution of this strategy will require an ongoing dialogue between clinicians, scientists, regulators and industry to take full advantage of advances in our understanding of the biology of these processes and their appropriate application to patients.

Whole-organ cardiac transplantation is the only currently available clinical means of replacing the lost muscle, but this option is limited by the inadequate supply of donor hearts. Thus, cell-based cardiac repair has attracted considerable interest as an alternative means of ameliorating cardiac injury. Because of their tremendous capacity for expansion and unquestioned cardiomyogenic potential, pluripotent human embryonic stem cells (hESCs) represent an attractive candidate cell source for deriving large quantities of cardiomyocytes for such therapies. To effectively repair the heart, however, there is a need for homogenous cardiomyocyte preparations as well as for improved cell survival after implantation. In this presentation, potential solutions to both challenges will be discussed.

First, we have found that a combination of guided in vitro differentiation and subsequent cardiac enrichment steps can produce suitable numbers of highly purified human cardiomyocytes. These cells express expected cardiac markers and exhibit mechanisms of excitation-contraction coupling remarkably similar to those of adult human cardiomyocytes. Second, we have developed a novel, combinatorial prosurvival “cocktail” (PSC) that, when delivered with cells into infarcted rats’ hearts, robustly improves graft outcome (increasing the engraftment rates from 18 percent to 100 percent for recipients at 4 weeks posttransplantation). Most importantly, analysis by echocardiography and magnetic resonance imaging indicated that the transplantation of hESC-derived cardiomyocytes in PSC preserved global and regional contractile function—effects not mediated by the PSC vehicle alone or by the noncardiac cell controls. These preclinical studies demonstrate that hESC-derived cardiomyocytes can mediate the remuscularization of infarcted hearts and that this intervention preserves ventricular function.

In addition to the need for the preclinical testing of safety and efficacy in a larger animal model with a slower heart rate, several significant challenges remain to the successful development of hESC-based cardiac cell therapies. The field must address the concerns of immune rejection, arrhythmogenesis, and tumor formation. A better mechanistic understanding of the observed beneficial effects on cardiac function is also needed. The progress and perspectives of ongoing work addressing these issues will be discussed, including studies with induced pluripotent stem cells, intravital imaging to demonstrate host-graft electromechanical integration, the pharmacological control of electrical phenotype, and the improved organization of hESC-derived cardiac implants with templated hydrogels and other tissue-engineering approaches.

Heart failure is the leading cause of death in the elderly, but whether this is the result of a primary aging myopathy dictated by the depletion of the cardiac progenitor cell (CPC) pool is unknown. We have identified that chronological age leads to telomeric shortening in CPCs, which by necessity generate a differentiated progeny that rapidly acquires the senescent phenotype conditioning organ aging. CPC aging is mediated by the attenuation of the IGF-1-IGF-1R and HGF-c-Met systems, which do not counteract any longer the CPC renin-angiotensin-system, resulting in cellular senescence, growth arrest, and apoptosis. However, the pulse-chase-BrdU-labeling-assay revealed that the senescent heart contains functionally competent CPCs, which have the properties of stem cells. This subset of telomerase-competent CPCs have long telomeres and, following activation, migrate to the regions of damage, generating a population of young cardiomyocytes and reversing partly the aging myopathy. The senescent heart phenotype and heart failure are corrected to some extent, leading to the prolongation of maximum lifespan.

Hematopoietic stem cell therapy (HST) is an established and efficacious approach to the management of patients with nonmalignant and malignant blood disorders. Moreover, HST is an investigational modality for diverse disorders, including muscular sclerosis and autoimmune disease. HST relies on the potency of hematopoietic stem cells (HSCs) to give rise to all blood cells, including all myeloid and lymphoid cell types. Work of the past 25 years has defined the surface markers of HSCs and downstream progenitors that permit the prospective purification of cells for transplantation and biological study. Intrinsic cell fate decisions of HSCs and progenitors are mediated by nuclear transcription factors, many of which are the subjects of chromosomal translocation or somatic mutation in hematopoietic malignancies. HSCs reside within niches within the bone marrow where complex cellular and soluble components control cellular homeostatsis. In the presentation, basic aspects of the hematopoietic system will be discussed, with an emphasis on those aspects that are relevant to cellular therapy in other organ systems..

Muscular dystrophies are inherited diseases in which skeletal and commonly cardiac muscles degenerate and are replaced to varying extents by new muscle and/or scar tissue. In skeletal muscle, regeneration predominates in the early stages, but in the more severe forms, such as Duchenne muscular dystrophy, this process begins to fail after a few years. The strong early regeneration has prompted numerous studies of the involvement of precursor or stem cells, with the idea of using these as vectors for the genetic correction of the disease and/or of augmenting the failing regenerative mechanism so as to favor the rebuilding of healthy muscle tissue.

Initial efforts focused on the satellite cell that is closely associated with the mature muscle fiber as the source of such precursor or stem cells. Transplantation studies have revealed that this anatomically defined class of cells contains an element that exhibits robust stem cell functions. More recently, other sources of myogenic stem cells have been investigated, mainly cells derived from bone marrow; fat; and most recently, from the microvascular bed. All sources have been shown to be capable of becoming involved with muscle regeneration, but in most cases the efficiency has been far too low to be of potential functional use.
 
The most intractable problem for cell-based therapies of skeletal muscle is that of delivery to the large and diffusely distributed mass of body musculature. For this purpose, only one cell type, the mesoangioblast, derived from the microvascular pericyte population, is supported by data showing any acceptable level of efficacy.

On the level of fundamental biology, considerable doubt exists as to the extent to which skeletal muscle is actually maintained by a physiologically relevant stem cell compartment. Most available data in humans suggest that there is only trivial turnover of mature skeletal muscle in normal individuals who undertake moderate exercise. This limited capacity for stem cell-based maintenance may prove to be one of the important obstacles to be overcome if cell therapies are to be applied to human muscle disease.

Mesenchymal stromal cells (MSCs) originally attracted attention because they were easily isolated from bone marrow by their adherence to tissue culture surfaces and by their ability to differentiate into bone, cartilage, and fat both in vitro and in vivo. Based on observations in a transgenic mouse model, an initial clinical trial was carried out in which MSCs were administered to children with severe osteogenesis imperfecta. Currently, large numbers of patients are enrolled in clinical trials with MSCs or related cells from bone marrow for such diseases as arthritis, graft-versus-host disease, Crohn’s disease, heart disease, and diabetes. However, several paradoxes have been encountered. First, the cells can produce functional improvements in several organs after intravenous administration even though most intravenously infused MSCs are trapped in the lung and are destroyed. Second, the cells frequently produce functional improvements in disease models even though there is little evidence of the long-term engraftment of the cells.

We recently demonstrated that although most intravenously administered MSCs are trapped in the lung, a subpopulation of small, rapidly proliferating MSCs escapes entrapment and engrafts in injured tissues. Also, data by us and others demonstrated that although MSCs can differentiate into a variety of cell phenotypes, differentiation or cell fusion is a minor event under most circumstances. Instead, the cells repair injured tissues by a variety of actions, including the enhancement of tissue-endogenous stem/progenitor cells and the modulation of early inflammatory and immune reactions that are invoked by tissue injury.

Supported in part by National Institutes of Health grants HL 073252, P40 RR 17447, and P01 HL 075161.

The use of stem cells in novel restorative therapies remains a holy grail for translational research. But how close are we to achieving such a goal? Recently, human neural stem cells have been isolated from both embryonic stem cells and fetal brain tissue. They can be expanded in culture without losing the potential to differentiate into both neurons and glia. Simply replacing lost or damaged neurons in the brain using such stem cells may be more complicated than first imagined. Large projection neurons that die in such diseases as Parkinson’s disease and amyotrophic lateral sclerosis (ALS) require not only replacement but also rewiring. A more immediate use of stem cells, however, might be the replacement of damaged astrocytes and the delivery of large therapeutic proteins directly to the brain. Recent studies have shown that in animal models of Parkinson’s disease and ALS, growth factors, such as GDNF, IGF-1, and VEGF, can have significant neuroprotective effects. In a recent clinical trial we have shown that GDNF is safe when delivered directly to the brain of patients with Parkinson’s disease who show some signs of improvement over time. As stem cells migrate and integrate into the damaged nervous system, produce astrocytes, and be genetically modified to release growth factors, they represent an ideal source of tissue for delivery. Furthermore, evidence is accumulating that the glial cells produced by human neural stem cells are themselves neuroprotective under certain conditions. Thus, combining the inherent trophic effects of glia derived from stem cells with selective gene therapy methods it may be possible to prevent or slow down cell death in certain neurological illnesses.

The liver and pancreas both arise from embryonic endoderm cells and share many other characteristics of early development. Yet each tissue originates from multiple domains of the endoderm, under the influence of different regulatory genes and inductive cues. The diversity of natural mechanisms for generating liver and pancreas progenitors is anticipated to allow flexibility in programming liver and pancreas cells (e.g., insulin cells) from other progenitor and stem cell types. Notably, liver and pancreas cell specification occurs in major phases that should be understood for prospective stem cell programming: competence, whereby pioneer transcription factors and epigenetic marks "prime" genes for potential activity; induction, whereby specific extracellular signals impinge on pioneer factors and other regulators to activate certain genetic programs while repressing others; and stabilization, whereby initial metastable differentiation states, which are reversible, are converted to stable states that are much more difficult to reverse. Each of these will be reviewed. Also, new studies reveal that the differences in the regenerative capacity of the adult liver and pancreas cells appear to be established at the embryonic progenitor stage. Understanding the basis for stable cell programming and intrinsic regenerative capacities should aid in the targeted programming and growth of stem cells.