Direct Conversion of Pluripotent Human Embryonic Stem Cells Under Defined Culture Conditions into Human Neuronal or Cardiomyocyte Cell Therapy Derivatives.

Developing novel strategies for well-controlled efficiently directing pluripotent human embryonic stem cells (hESCs) exclusively and uniformly towards clinically relevant cell types in a lineage-specific manner is not only crucial for unveiling the molecular and cellular cues that direct human embryogenesis but also vital to harnessing the power of hESC biology for tissue engineering and cell-based therapies. Conventional hESC differentiation methods require uncontrollable simultaneous multi-lineage differentiation of pluripotent cells, which yield embryoid bodies (EB) or aggregates consisting of a mixed population of cell types of three embryonic germ layers, among which only a very small fraction of cells display targeted differentiation, impractical for commercial and clinical applications. Here, a protocol for lineage-specific differentiation of hESCs, maintained under defined culture systems, direct from the pluripotent stage using small-molecule induction exclusively and uniformly to a neural or a cardiac lineage is described. Lineage-specific differentiation of pluripotent hESCs by small-molecule induction enables well-controlled highly efficient direct conversion of nonfunctional pluripotent hESCs into a large supply of high-purity functional human neuronal or cardiomyocyte cell therapy derivatives for commercial and therapeutic uses.

Human Stem Cell Derivatives Retain More Open Epigenomic Landscape When Derived from Pluripotent Cells than from Tissues.

The growing number of identified stem cell derivatives and escalating concerns for safety and efficacy of these cells towards clinical applications have made it increasingly crucial to be able to assess the relative risk-benefit ratio of a given stem cell from a given source for a particular disease. Discerning the intrinsic plasticity and regenerative potential of human stem cell populations might reside in chromatin modifications that shape the respective epigenomes of their derivation routes. Previously, we have generated engraftable human neuronal progenitors direct from pluripotent human embryonic stem cells (hESCs) by small molecule induction (hESC-I hNuPs). Unlike the prototypical neuroepithelial-like nestin-positive human neural stem cells (hNSCs), these in vitro neuroectoderm-derived Nurr1-positive hESC-I hNuPs are a more neuronal lineage-specific and plastic hESC derivative. In this study, the global chromatin landscape changes in pluripotent hESCs and their neuronal lineage-specific derivative hESC-I hNuPs were profiled using genome-wide mapping and compared to CNS tissue-derived hNSCs. This study found that the broad potential of pluripotent hESCs is defined by an epigenome constituted of open conformation of chromatin mediated by a pattern of Oct-4 global distribution that corresponds closely with those of acetylated nucleosomes genome-wide. The epigenomic transition from pluripotency to restriction in lineage choices is characterized by genome-wide increases in histone H3K9 methylation that mediates global chromatin-silencing and somatic identity. Tissue-resident CNS-derived hNSCs have acquired a substantial number of additional histone H3K9 methylation, therefore, more silenced chromatin. These data suggest that the intrinsic plasticity and regenerative potential of human stem cell derivatives can be differentiated by their epigenomic landscape features, and that human stem cell derivatives retain more open epigenomic landscape, therefore, more developmental potential for scale-up regeneration, when derived from the hESCs in vitro than from the CNS tissue in vivo.

Embedding the Future of Regenerative Medicine into the Open Epigenomic Landscape of Pluripotent Human Embryonic Stem Cells.

It has been recognized that pluripotent human embryonic stem cells (hESCs) must be transformed into fate-restricted derivatives before use for cell therapy. Realizing the therapeutic potential of pluripotent hESC derivatives demands a better understanding of how a pluripotent cell becomes progressively constrained in its fate options to the lineages of tissue or organ in need of repair. Discerning the intrinsic plasticity and regenerative potential of human stem cell populations reside in chromatin modifications that shape the respective epigenomes of their derivation routes. The broad potential of pluripotent hESCs is defined by an epigenome constituted of open conformation of chromatin mediated by a pattern of Oct-4 global distribution that corresponds genome-wide closely with those of active chroma tin modifications. Dynamic alterations in chromatin states correlate with loss-of-Oct4-associated hESC differentiation. The epigenomic transition from pluripotence to restriction in lineage choices is characterized by genome-wide increases in histone H3K9 methylation that mediates global chromatin-silencing and somatic identity. Human stem cell derivatives retain more open epigenomic landscape, therefore, more developmental potential for scale-up regeneration, when derived from the hESCs in vitrothan from the CNS tissuein vivo . Recent technology breakthrough enables direct conversion of pluripotent hESCs by small molecule induction into a large supply of lineage-specific neuronal cells or heart muscle cells with adequate capacity to regenerate neurons and contractile heart muscles for developing safe and effective stem cell therapies. Nuclear translocation of NAD-dependent histone deacetylase SIRT1 and global chromatin silencing lead to hESC cardiac fate determination, while silencing of pluripotence-associated hsa-miR-302 family and drastic up-regulation of neuroectodermal Hox miRNA hsa-miR-10 family lead to hESC neural fate determination. These recent studies place global chromatin dynamics as central to tracking the normal pluripotence and lineage progres sion of hESCs. Embedding lineage-specific genetic and epigenetic developmental programs into the open epigenomic landscape of pluripotent hESCs offers a new repository of human stem cell therapy derivatives for the future of regenerative medicine.

Constraining the Pluripotent Fate of Human Embryonic Stem Cells for Tissue Engineering and Cell Therapy - The Turning Point of Cell-Based Regenerative Medicine.

To date, the lack of a clinically-suitable source of engraftable human stem/progenitor cells with adequate neurogenic potential has been the major setback in developing safe and effective cell-based therapies for regenerating the damaged or lost CNS structure and circuitry in a wide range of neurological disorders. Similarly, the lack of a clinically-suitable human cardiomyocyte source with adequate myocardium regenerative potential has been the major setback in regenerating the damaged human heart. Given the limited capacity of the CNS and heart for self-repair, there is a large unmet healthcare need to develop stem cell therapies to provide optimal regeneration and reconstruction treatment options to restore normal tissues and function. Derivation of human embryonic stem cells (hESCs) provides a powerful in vitro model system to investigate molecular controls in human embryogenesis as well as an unlimited source to generate the diversity of human somatic cell types for regenerative medicine. However, realizing the developmental and therapeutic potential of hESC derivatives has been hindered by the inefficiency and instability of generating clinically-relevant functional cells from pluripotent cells through conventional uncontrollable and incomplete multi-lineage differentiation. Recent advances and breakthroughs in hESC research have overcome some major obstacles in bringing hESC therapy derivatives towards clinical applications, including establishing defined culture systems for de novo derivation and maintenance of clinical-grade pluripotent hESCs and lineage-specific differentiation of pluripotent hESCs by small molecule induction. Retinoic acid was identified as sufficient to induce the specification of neuroectoderm direct from the pluripotent state of hESCs and trigger a cascade of neuronal lineage-specific progression to human neuronal progenitors and neurons of the developing CNS in high efficiency, purity, and neuronal lineage specificity by promoting nuclear translocation of the neuronal specific transcription factor Nurr-1. Similarly, nicotinamide was rendered sufficient to induce the specification of cardiomesoderm direct from the pluripotent state of hESCs by promoting the expression of the earliest cardiac-specific transcription factor Csx/Nkx2.5 and triggering progression to cardiac precursors and beating cardiomyocytes with high efficiency. This technology breakthrough enables direct conversion of pluripotent hESCs into a large supply of high purity neuronal cells or heart muscle cells with adequate capacity to regenerate CNS neurons and contractile heart muscles for developing safe and effective stem cell therapies. Transforming pluripotent hESCs into fate-restricted therapy derivatives dramatically increases the clinical efficacy of graft-dependent repair and safety of hESC-derived cellular products. Such milestone advances and medical innovations in hESC research allow generation of a large supply of clinical-grade hESC therapy derivatives targeting for major health problems, bringing cell-based regenerative medicine to a turning point.

Defining Conditions for Sustaining Epiblast Pluripotence Enables Direct Induction of Clinically-Suitable Human Myocardial Grafts from Biologics-Free Human Embryonic Stem Cells.

To date, lacking of a clinically-suitable human cardiac cell source with adequate myocardium regenerative potential has been the major setback in regenerating the damaged human myocardium. Pluripotent Human Embryonic Stem Cells (hESCs) proffer unique revenue to generate a large supply of cardiac lineage-committed cells as human myocardial grafts for cell-based therapy. Due to the prevalence of heart disease worldwide and acute shortage of donor organs or human myocardial grafts, there is intense interest in developing hESC-based therapy for heart disease and failure. However, realizing the potential of hESCs has been hindered by the inefficiency and instability of generating cardiac cells from pluripotent cells through uncontrollable multi-lineage differentiation. In addition, the need for foreign biologics for derivation, maintenance, and differentiation of hESCs may make direct use of such cells and their derivatives in patients problematic. Understanding the requirements for sustaining pluripotentce and self-renewal of hESCs will provide the foundation for de novo derivation and long-term maintenance of biologics-free hESCs under optimal yet well-defined culture conditions from which they can be efficiently directed towards clinically-relevant lineages for therapies. We previously reported the resolving of the elements of a defined culture system, serving as a platform for effectively directing pluripotent hESCs uniformly towards a cardiac lineage-specific fate by small molecule induction. In this study, we found that, under the defined culture conditions, primitive endoderm-like (PEL) cells constitutively emerged and acted through the activin-A-SMAD pathway in a paracrine fashion to sustain the epiblast pluripotence of hESCs. Such defined conditions enable the spontaneous unfolding of inherent early embryogenesis processes that, in turn, aid efficient clonal propagation and de novo derivation of stable biologics-free hESCs from blastocysts that can be directly differentiated into a large supply of clinically-suitable human myocardial grafts across the spectrum of developmental stages using small molecule induction for cardiovascular repair.

MicroRNA Profiling Reveals Distinct Mechanisms Governing Cardiac and Neural Lineage-Specification of Pluripotent Human Embryonic Stem Cells.

Realizing the potential of human embryonic stem cells (hESCs) has been hindered by the inefficiency and instability of generating desired cell types from pluripotent cells through multi-lineage differentiation. We recently reported that pluripotent hESCs maintained under a defined platform can be uniformly converted into a cardiac or neural lineage by small molecule induction, which enables lineage-specific differentiation direct from the pluripotent state of hESCs and opens the door to investigate human embryonic development using in vitro cellular model systems. To identify mechanisms of small molecule induced lineage-specification of pluripotent hESCs, in this study, we compared the expression and intracellular distribution patterns of a set of cardinal chromatin modifiers in pluripotent hESCs, nicotinamide (NAM)-induced cardiomesodermal cells, and retinoic acid (RA)-induced neuroectodermal cells. Further, genome-scale profiling of microRNA (miRNA) differential expression patterns was used to monitor the regulatory networks of the entire genome and identify the development-initiating miRNAs in hESC cardiac and neural lineage-specification. We found that NAM induced nuclear translocation of NAD-dependent histone deacetylase SIRT1 and global chromatin silencing, while RA induced silencing of pluripotence-associated hsa-miR-302 family and drastic up-regulation of neuroectodermal Hox miRNA hsa-miR-10 family to high levels. Genome-scale miRNA profiling indentified that a unique set of pluripotence-associated miRNAs was down-regulated, while novel sets of distinct cardiac- and neural-driving miRNAs were up-regulated upon the induction of lineage-specification direct from the pluripotent state of hESCs. These findings suggest that a predominant epigenetic mechanism via SIRT1-mediated global chromatin silencing governs NAM-induced hESC cardiac fate determination, while a predominant genetic mechanism via silencing of pluripotence-associated hsa-miR-302 family and drastic up-regulation of neuroectodermal Hox miRNA hsa-miR-10 family governs RA-induced hESC neural fate determination. This study provides critical insight into the earliest events in human embryogenesis as well as offers means for small molecule-mediated direct control and modulation of hESC pluripotent fate when deriving clinically-relevant lineages for regenerative therapies.

An Engraftable Human Embryonic Stem Cell Neuronal Lineage-Specific Derivative Retains Embryonic Chromatin Plasticity for Scale-Up CNS Regeneration.

BACKGROUND: Pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for repair. However, realizing the therapeutic potential of hESC derivatives has been hindered by generating neuronal cells from pluripotent cells through uncontrollable and inefficient multi-lineage differentiation. Previously, we used a defined platform to identify retinoic acid as sufficient to induce the specification of neuroectoderm direct from the pluripotent state of hESCs and trigger uniform neuronal lineage-specific progression to human neuronal progenitors (hESC-I hNuPs) and neurons (hESC-I hNus) in the developing CNS with high efficiency. METHODS: Having achieved uniformly conversion of pluripotent hESCs to a neuronal lineage, in this study, the expression and intracellular distribution patterns of a set of chromatin modifiers in hESC-I hNuPs were examined and compared to the two prototypical neuroepithelial-like human neural stem cells (hNSCs) either derived from hESCs or isolated directly from the human fetal neuroectoderm in vivo. RESULTS: These hESC-I hNuPs expressed high levels of active chromatin modifiers, including acetylated histone H3 and H4, HDAC1, Brg-1, and hSNF2H, retaining an embryonic acetylated globally active chromatin state. Consistent with this observation, several repressive chromatin remodeling factors regulating histone H3K9 methylation, including SIRT1, SUV39H1, and Brm, were inactive in hESC-I hNuPs. These Nurr1-positive hESC-I hNuPs, which did not express the canonical hNSC markers, yielded neurons efficiently and exclusively, as they did not differentiate into glial cells. Following engraftment in the brain, hESC-I hNuPs yielded well-dispersed and well-integrated human neurons at a high prevalence. CONCLUSIONS: These observations suggest that, unlike the prototypical neuroepithelial-like nestin-positive hNSCs, these in vitro neuroectoderm-derived Nurr1-positive hESC-I hNuPs are a more neuronal lineage-specific and plastic human stem cell derivative, providing an engraftable human embryonic neuronal progenitor in high purity and large supply with adequate neurogenic potential for scale-up CNS regeneration as stem cell therapy to be translated to patients in clinical trials.

The Dynamics of Global Chromatin Remodeling are Pivotal for Tracking the Normal Pluripotency of Human Embryonic Stem Cells.

Pluripotent Human Embryonic Stem Cells (hESCs) have the unconstrained capacity for long-term stable undifferentiated growth in culture and unrestricted developmental capacity. Packaging of the eukaryotic genome into chromatin confers higher order structural control over maintaining stem cell plasticity and directing differentiation. We recently reported the establishment of a defined culture system for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards functional lineages. To unveil the epigenetic mechanism in maintaining the epiblast pluripotence of hESCs, in this study, the global chromatin dynamics in the pluripotent hESCs maintained under the defined culture were examined. This study shows that the genomic plasticity of pluripotent hESCs is enabled by an acetylated globally active chromatin maintained by Oct-4. The pluripotency of hESCs that display normal stable expansion is associated with high levels of expression and nuclear localization of active chromatin remodeling factors that include acetylated histone H3 and H4, Brg-1, hSNF2H, HAT p300, and HDAC1; weak expression or cytoplasmic localization of repressive chromatin remodeling factors that are implicated in transcriptional silencing; and residual H3 K9 methylation. A dynamic progression from acetylated to transient hyperacetylated to hypoacetylated chromatin states correlates with loss-of-Oct4-associated hESC differentiation. RNA interference directed against Oct-4 and HDAC inhibitor analysis support this pivotal link between chromatin dynamics and hESC differentiation. These findings reveal an epigenetic mechanism for placing global chromatin dynamics as central to tracking the normal pluripotency and lineage progression of hESCs.

Genome-Scale Mapping of MicroRNA Signatures in Human Embryonic Stem Cell Neurogenesis.

To date, lacking of a clinically-suitable source of engraftable human stem/progenitor cells with adequate neurogenic potential has been the major setback in developing effective cell-based therapies against a wide range of neurological disorders. Derivation of human embryonic stem cells (hESCs) provides a powerful tool to investigate the molecular controls in human embryonic neurogenesis as well as an unlimited source to generate the diversity of human neuronal cell types in the developing CNS for repair. However, realizing the developmental and therapeutic potential of hESCs has been hindered by conventional multi-lineage differentiation of pluripotent cells, which is uncontrollable, inefficient, highly variable, difficult to reproduce and scale-up. We recently identified retinoic acid (RA) as sufficient to induce the specification of neuroectoderm direct from the pluripotent state of hESCs under defined platform and trigger progression to human neuronal progenitors (hESC-I hNuPs) and neurons (hESC-I hNus) in the developing CNS with high efficiency, which enables hESC neuronal lineage-specific differentiation and opens the door to investigate human embryonic neurogenesis using the hESC model system. In this study, genome-scale profiling of microRNA (miRNA) differential expression patterns in hESC neuronal lineage-specific progression was used to identify molecular signatures of human embryonic neurogenesis. These in vitro neuroectoderm-derived human neuronal cells have acquired a neuron al identity by down-regulating pluripotence-associated miRNAs and inducing the expression of miRNAs linked to regulating human CNS development to high levels in a stage-specific manner, including silencing of the prominent pluripotence-associated hsa-miR-302 family and drastic expression increases of the Hox hsa-miR-10 and let-7 miRNAs. Following transplantation, hESC-I hNuPs engrafted and yielded well-integrated neurons at a high prevalence within neurogenic regions of the brain. In 3D culture, these hESC-I hNuPs proceeded to express subtype neuronal markers, such as dopaminergic and motor neurons, demonstrating their therapeutic potential for CNS repair. Our study provides critical insight into molecular neurogenesis in human embryonic development as well as offers an adequate human neurogenic cell source in high purity and large quantity for scale-up CNS regeneration.

Efficient derivation of human neuronal progenitors and neurons from pluripotent human embryonic stem cells with small molecule induction.

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging(1-3). Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells(1-3). Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration(1,4-7). However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity(7-10). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic(11-13). To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules(14) (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders(1, 14). And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons(1, 14, 15). However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

Efficient derivation of human cardiac precursors and cardiomyocytes from pluripotent human embryonic stem cells with small molecule induction.

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

Patents on Technologies of Human Tissue and Organ Regeneration from Pluripotent Human Embryonic Stem Cells.

Human embryonic stem cells (hESCs) are genetically stable with unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of a large supply of disease-targeted human somatic cells that are restricted to the lineage in need of repair. There is a large healthcare need to develop hESC-based therapeutic solutions to provide optimal regeneration and reconstruction treatment options for the damaged or lost tissue or organ that have been lacking. In spite of controversy surrounding the ownership of hESCs, the number of patent applications related to hESCs is growing rapidly. This review gives an overview of different patent applications on technologies of derivation, maintenance, differentiation, and manipulation of hESCs for therapies. Many of the published patent applications have been based on previously established methods in the animal systems and multi-lineage inclination of pluripotent cells through spontaneous germ-layer differentiation. Innovative human stem cell technologies that are safe and effective for human tissue and organ regeneration in the clinical setting remain to be developed. Our overall view on the current patent situation of hESC technologies suggests a trend towards hESC patent filings on novel therapeutic strategies of direct control and modulation of hESC pluripotent fate, particularly in a 3-dimensional context, when deriving clinically-relevant lineages for regenerative therapies.

Important precautions when deriving patient-specific neural elements from pluripotent cells.

Multipotent human neural stem cells (hNSC) have traditionally been isolated directly from the central nervous system (CNS). To date, as a therapeutic tool in the treatment of neurologic disorders, the most promising results have been obtained using hNSC isolated directly from the human fetal neuroectoderm. The propagation ability of such tissue-derived hNSC is often limited, however, making it difficult to establish a large-scale culture. Following engraftment, these hNSC often show low efficiency in generating the desired neuronal cells necessary for reconstruction of the damaged host milieu and, as a result, have failed to give satisfactory results in clinical trials so far. Alternatively, human embryonic stem cells (hESC) offer a pluripotent reservoir for in vitro derivation of a rich spectrum of well-characterized neural-lineage committed stem/progenitor/precursor cells that can, theoretically, be picked at precisely their safest and most efficacious state of plasticity to meet a given clinical challenge. However, the need for 'foreign' biologic additives and multilineage differentiation inclination may make direct use of such cell-derived hNSC in patients problematic. The hNSC, when derived from pluripotent cells under protocols presently employed in the field, tend to display not only a low efficiency in neuronal differentiation, but also an inclination for phenotypic heterogeneity and instability and, hence, increased risk of tumorigenesis following engraftment. For hNSC derived in vitro to be used safely in therapeutic paradigms, it requires conversion of human pluripotent cells uniformly to cells that are restricted to the neural lineage in need of repair. Developing strategies for direct induction of human pluripotent cells exclusively into neural-committed progenies at a broad range of developmental stages will allow a large supply of optimal therapeutic hNSC tailor-made for safe and effective treatment of particular neurologic diseases and injuries in patients.

Behavioral improvement in a primate Parkinson's model is associated with multiple homeostatic effects of human neural stem cells.

Stem cells have been widely assumed to be capable of replacing lost or damaged cells in a number of diseases, including Parkinson's disease (PD), in which neurons of the substantia nigra (SN) die and fail to provide the neurotransmitter, dopamine (DA), to the striatum. We report that undifferentiated human neural stem cells (hNSCs) implanted into 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated Parkinsonian primates survived, migrated, and had a functional impact as assessed quantitatively by behavioral improvement in this DA-deficit model, in which Parkinsonian signs directly correlate to reduced DA levels. A small number of hNSC progeny differentiated into tyrosine hydroxylase (TH) and/or dopamine transporter (DAT) immunopositive cells, suggesting that the microenvironment within and around the lesioned adult host SN still permits development of a DA phenotype by responsive progenitor cells. A much larger number of hNSC-derived cells that did not express neuronal or DA markers was found arrayed along the persisting nigrostriatal path, juxtaposed with host cells. These hNSCs, which express DA-protective factors, were therefore well positioned to influence host TH+ cells and mediate other homeostatic adjustments, as reflected in a return to baseline endogenous neuronal number-to-size ratios, preservation of extant host nigrostriatal circuitry, and a normalizing effect on alpha-synuclein aggregation. We propose that multiple modes of reciprocal interaction between exogenous hNSCs and the pathological host milieu underlie the functional improvement observed in this model of PD.