Recent advances in human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) biology enable generation of dopaminergic neurons for potential therapy and drug screening. However, our current understanding of molecular and cellular signaling that controls human dopaminergic development and function is limited. Here, we report on a whole genome analysis of gene expression during dopaminergic differentiation of human ESC/iPSC using Illumina bead microarrays. We generated a transcriptome data set containing the expression levels of 28,688 unique transcripts by profiling five lines (three ESC and two iPSC lines) at four stages of differentiation: (1) undifferentiated ESC/iPSC, (2) neural stem cells, (3) dopaminergic precursors, and (4) dopaminergic neurons. This data set provides comprehensive information about genes expressed at each stage of differentiation. Our data indicate that distinct pathways are activated during neural and dopaminergic neuronal differentiation. For example, WNT, sonic hedgehog (SHH), and cAMP signaling pathways were found over-represented in dopaminergic populations by gene enrichment and pathway analysis, and their role was confirmed by perturbation analyses using RNAi (small interfering RNA of SHH and WNT) or small molecule [dibutyryl cyclic AMP (dcAMP)]. In summary, whole genome profiling of dopaminergic differentiation enables systematic analysis of genes/pathways, networks, and cellular/molecular processes that control cell fate decisions. Such analyses will serve as the foundation for better understanding of dopaminergic development, function, and development of future stem cell-based therapies.
The authors describe a pair of transcription activator-like effector nucleases (TALENs) that enable more efficient genome editing than the commercially available zinc finger nuclease at the AAVS1 site. Results show that high-efficiency targeting can be obtained with open-source TALENs and that careful optimization of the reporter and transgene constructs results in stable and persistent expression in vitro and in vivo.
Human induced pluripotent stem (hiPS) cell lines with tissue-specific or ubiquitous reporter genes are extremely useful for optimizing in vitro differentiation conditions as well as for monitoring transplanted cells in vivo. The adeno-associated virus integration site 1 (AAVS1) locus has been used as a “safe harbor” locus for inserting transgenes because of its open chromatin structure, which permits transgene expression without insertional mutagenesis. However, it is not clear whether targeted transgene expression at the AAVS1 locus is always protected from silencing when driven by various promoters, especially after differentiation and transplantation from hiPS cells. In this paper, we describe a pair of transcription activator-like effector nucleases (TALENs) that enable more efficient genome editing than the commercially available zinc finger nuclease at the AAVS1 site. Using these TALENs for targeted gene addition, we find that the cytomegalovirus-immediate early enhancer/chicken β-actin/rabbit β-globin (CAG) promoter is better than cytomegalovirus 7 and elongation factor 1α short promoters in driving strong expression of the transgene. The two independent AAVS1, CAG, and enhanced green fluorescent protein (EGFP) hiPS cell reporter lines that we have developed do not show silencing of EGFP either in undifferentiated hiPS cells or in randomly and lineage-specifically differentiated cells or in teratomas. Transplanting cardiomyocytes from an engineered AAVS1-CAG-EGFP hiPS cell line in a myocardial infarcted mouse model showed persistent expression of the transgene for at least 7 weeks in vivo. Our results show that high-efficiency targeting can be obtained with open-source TALENs and that careful optimization of the reporter and transgene constructs results in stable and persistent expression in vitro and in vivo.
Genome editing; Human induced pluripotent stem cells; Transplantation; Transcription activator-like effector nuclease (TALEN); AAVS1; Differentiation
There is growing recognition of the potential value of human induced pluripotent stem cells (hiPSC) for understanding disease and identifying drugs targets. This has been reflected in the establishment of multiple large-scale hiPSC initiatives worldwide. Representatives of these met recently at a workshop supported by the Welcome Trust in the UK and in a focus session at the 2014 ISSCR annual meeting in Vancouver. The purpose was to discuss strategies for making thousands of hiPSC lines widely available with as few restrictions as possible while retaining financial viability and donor privacy. The outcome of these discussions is described here.
The field of regenerative medicine has been revolutionized by breakthroughs in stem cell biology, gene engineering, and whole-genome sequencing. These advances are not only scientific or medical but have also advanced how we conceptualize regenerative medicine. The progenitive research that proceeded as well as a substantial part of the funding that supported these discoveries were provided by the National Institutes of Health (NIH). Now, perhaps more than ever, the NIH has a vital role to play in the translation of science into clinical practice. The NIH is uniquely positioned to coordinate interactions between the different institutes and other arms of the government, as well as international organizations. Efforts of researchers in the United States both within and without the NIH are supported by a number of mechanisms, including specialized workshops, and the support of developing small-scale industry. Additionally, the NIH has stepped up to provide necessary infrastructure in areas of regenerative medicine where the medical need might be apparent but might be currently infeasible or unattractive to private-sector investment. This article will discuss these perhaps lesser-known activities of the NIH, which I believe have continued and will continue to contribute to the role of stem cell research in translating science into regenerative medicine.
This study developed a highly efficient serum-free pluripotent stem cell (PSC) neural induction medium that can induce human PSCs into primitive neural stem cells (NSCs) in 7 days, obviating the need for time-consuming, laborious embryoid body generation or rosette picking. This method of primitive NSC derivation sets the stage for the scalable production of clinically relevant neural cells for cell therapy applications in good manufacturing practice conditions.
Human pluripotent stem cells (hPSCs), including human embryonic stem cells and human induced pluripotent stem cells, are unique cell sources for disease modeling, drug discovery screens, and cell therapy applications. The first step in producing neural lineages from hPSCs is the generation of neural stem cells (NSCs). Current methods of NSC derivation involve the time-consuming, labor-intensive steps of an embryoid body generation or coculture with stromal cell lines that result in low-efficiency derivation of NSCs. In this study, we report a highly efficient serum-free pluripotent stem cell neural induction medium that can induce hPSCs into primitive NSCs (pNSCs) in 7 days, obviating the need for time-consuming, laborious embryoid body generation or rosette picking. The pNSCs expressed the neural stem cell markers Pax6, Sox1, Sox2, and Nestin; were negative for Oct4; could be expanded for multiple passages; and could be differentiated into neurons, astrocytes, and oligodendrocytes, in addition to the brain region-specific neuronal subtypes GABAergic, dopaminergic, and motor neurons. Global gene expression of the transcripts of pNSCs was comparable to that of rosette-derived and human fetal-derived NSCs. This work demonstrates an efficient method to generate expandable pNSCs, which can be further differentiated into central nervous system neurons and glia with temporal, spatial, and positional cues of brain regional heterogeneity. This method of pNSC derivation sets the stage for the scalable production of clinically relevant neural cells for cell therapy applications in good manufacturing practice conditions.
Astrocytes; Cell culture; Neural stem cell; Neural induction; Neural differentiation; Oligodendrocytes; Neuron; Nestin
Bone morphogenetic proteins (BMPs), a group of cytokines in the TGF-β superfamily, have complex regulatory roles in the control of neural proliferation and cell fate decision. In this study, we analyzed the potential role(s) of BMP signaling on the regulation of the proliferation and differentiation of the unique progenitor cells of the neonatal anterior subventricular zone (SVZa). Unlike other progenitor cells of the brain, SVZa progenitor cells have the capacity to divide even though they express a neuronal phenotype. In order to augment or inhibit endogenous BMP signaling, we injected into the neonatal rat SVZa replication-deficient retroviruses encoding for either the wild-type BMP receptor subtype Ia (wt-BMPR-Ia) or a mutated dominant-negative version of BMPR-Ia (dn-BMPR-Ia) in conjunction with a reporter gene, human alkaline phosphatase (AP) and perfused the pups 1, 4 and 7 days post injection. We analyzed whether changing the expression of BMPR-Ia has an effect on the spatial-temporal expression pattern of the cyclin dependent kinase inhibitor, p19INK4d, or on the phenotype of SVZa derived cells. The results of our study confirmed and extended our previous findings that in control (non injected) animals, the rostral migratory stream (RMS), traversed by the SVZa-derived cells en route to the olfactory bulb, exhibits an anteriorhigh-posteriorlow gradient of p19INK4d expression; p19INK4d expression is essentially absent in the SVZa and highest in the subependymal zone in the middle of the olfactory bulb. However, SVZa progenitor cells encoding the wt-BMPR-Ia gene express p19INK4d within the SVZa, suggesting that the BMPs induce SVZa cells to ectopically undergo cell cycle exit within the SVZa. Furthermore, unlike striatal SVZ progenitor cells, which acquire an astrocytic phenotype when exposed to BMPs, SVZa progenitor cells retain their neuronal commitment under augmented BMP signaling.
Bone morphogenetic proteins; Cell cycle; p19INK4d; Progenitor cells; Retrovirus; Rostral migratory stream; Subverticular zone
In a screen for novel cell surface markers of neuronal progenitors, we recently identified mAb 2F7 that recognizes an epitope present on both progenitor cells and postmitotic neurons, in the developing CNS and PNS. In the embryonic rat telencephalon, the mAb 2F7 epitope is expressed by migratory and postmigratory neurons in the developing cerebral cortex, as well as by presumptive neuronal progenitor cells of the ventricular zone. In the neonatal forebrain mAb 2F7 labels postmitotic neurons, including those of the developing cerebral cortex and olfactory bulb, as well as the axons of the corpus callosum. While mAb 2F7 immunoreactivity is present on only a low density of the neuronal progenitor cells situated in the anterior part of the subventricular zone, a progressively higher proportion of cells forming the rostral migratory stream express this epitope. mAb 2F7 labels the surfaces of neurons and neuronal precursors, but not mature oligodendrocytes and astrocytes in primary cultures derived from the rat neural tube. In vivo, migrating neural crest cells, motor neurons, and axonal projections associated with the spinal cord express the mAb 2F7 epitope. Immunoblot analyses reveal that the mAb 2F7 epitope resides on several high-molecular-weight, membrane-associated proteins, and is likely to be composed of N-linked carbohydrate. These findings suggest that mAb 2F7 recognizes a novel epitope that is present on progenitor cells and postmitotic, differentiating neurons in the developing mammalian nervous system.
Neuronal progenitor; Neuronal differentiation; Spinal cord; Forebrain; E-NCAM; HNK-1
This article presents a draft of Points to Consider when using human somatic cells obtained from research donors to derive and subsequently distribute induced pluripotent stem cells (iPSCs), with the goal of initiating a deliberative process to develop consensus for such use.
Human somatic cell reprogramming is a leading technology for accelerating disease modeling and drug discovery. Research organizations are sponsoring initiatives to create libraries of induced pluripotent stem cell (iPSC) lines for broad distribution and application. Donor informed consent plays a critical role in supporting the ethical conduct of iPSC research. To date, our organizations have focused on informed consent considerations for somatic cell collection intended specifically for iPSC derivation and distribution. This article considers how somatic cells obtained under general (biomedical) research protocols can be used for iPSC derivation. We present draft Points to Consider regarding the use of human somatic cells for iPSC research. Our goal is to initiate a process designed to develop consensus for the use of previously collected specimens for iPSC research. We anticipate publishing final considerations in early 2014.
The ability to reprogram somatic cells to induced pluripotent stem cells (iPSCs) offers an opportunity to generate pluripotent patient-specific cell lines that can help model human diseases. These iPSC lines could also be powerful tools for drug discovery and the development of cellular transplantation therapies. Many methods exist for generating iPSC lines but those best suited for use in studying human diseases and developing therapies must be of adequate efficiency to produce iPSCs from samples that may be of limited abundance, capable of reprogramming cells from both skin fibroblasts and blood, and footprint-free. Several reprogramming techniques meet these criteria and can be utilized to derive iPSCs in projects with both basic scientific and therapeutic goals. Combining these reprogramming methods with small molecule modulators of signaling pathways can lead to successful generation of iPSCs from even the most recalcitrant patient-derived somatic cells.
Induced pluripotent stem cells; Reprogramming human somatic cells; Footprint-free iPSCs; Reprogramming with small molecules
Using India as a model system, this study reviews the role of key agencies and processes involved in the field of stem cell-based clinical research. This study identifies areas that need attention and provides solutions from other established and functioning models in the world to streamline and unify the regulatory and ethics approval processes for cell-based therapies; it also provides recommendations to check the growth and functioning of clinics offering unproven treatments.
Increasingly, a number of rapidly developing countries, including India, China, Brazil, and others, are becoming global hot spots for the development of regenerative medicine applications, including stem cell-based therapies. Identifying and overcoming regulatory and translational research challenges and promoting scientific and ethical clinical trials with cells will help curb the growth of stem cell tourism for unproven therapies. It will also enable academic investigators, local regulators, and national and international biotechnology and biopharmaceutical companies to accelerate stem cell-based clinical research that could lead to effective innovative treatments in these regions. Using India as a model system and obtaining input from regulators, clinicians, academics, and industry representatives across the stem cell field in India, we reviewed the role of key agencies and processes involved in this field. We have identified areas that need attention and here provide solutions from other established and functioning models in the world to streamline and unify the regulatory and ethics approval processes for cell-based therapies. We also make recommendations to check the growth and functioning of clinics offering unproven treatments. Addressing these issues will remove considerable hurdles to both local and international investigators, accelerate the pace of research and development, and create a quality environment for reliable products to emerge. By doing so, these countries would have taken one important step to move to the forefront of stem cell-based therapeutics.
Cellular therapy; Clinical trials; Stem cells; Clinical translation
Astrocytes are the most abundant cell type in the central nervous system (CNS) and have a multitude of functions that include maintenance of CNS homeostasis, trophic support of neurons, detoxification, and immune surveillance. It has only recently been appreciated that astrocyte dysfunction is a primary cause of many neurological disorders. Despite their importance in disease very little is known about global gene expression for human astrocytes. We have performed a microarray expression analysis of human fetal astrocytes to identify genes and signaling pathways that are important for astrocyte development and maintenance. Our analysis confirmed that the fetal astrocytes express high levels of the core astrocyte marker GFAP and the transcription factors from the NFI family which have been shown to play important roles in astrocyte development. A group of novel markers were identified that distinguish fetal astrocytes from pluripotent stem cell-derived neural stem cells (NSCs) and NSC-derived neurons. As in murine astrocytes, the Notch signaling pathway appears to be particularly important for cell fate decisions between the astrocyte and neuronal lineages in human astrocytes. These findings unveil the repertoire of genes expressed in human astrocytes and serve as a basis for further studies to better understand astrocyte biology, especially as it relates to disease.
The mesenchymal stromal cell (MSC) field continues to rapidly progress with a number of clinical trials initiated and completed, with some reported successes in multiple clinical indications, and a growing number of companies established. The field, nevertheless, faces several challenges. Persistent issues include the definition of a MSC and comparability between MSC preparations. This is because of inherent cell heterogeneity, the absence of markers that are unique to MSCs, and the difficulty in precisely defining them by developmental origin. Differences in the properties of MSCs also depend on the site of tissue harvest, phenotypic and genotypic characteristics of the donor and the isolation, and storage and expansion methods used. These differences may be sufficient to ensure that attributes of the final MSC product could differ in potentially significant ways. Since there are currently no gold standards, we propose using a reference material to establish methods of comparability among MSC preparations. We suggest four possible “ruler scenarios” and a method for global distribution. We further suggest that critical to establishing a reference material is the need to define protocols for comparing cells. The main purpose of this article is to solicit input in establishing a consensus-based comparison. A comparative approach will be critical to all stages of translation to better clarify mechanisms of MSC actions, define an optimal cell manufacturing process, ensure best practice clinical investigations, extend the use of an MSC product for new indications, protect an MSC product from imitators, and develop uniform reimbursement policies. Importantly, a reference material may enable a consensus on a practical definition of MSCs.
Recently, the U.S. Food and Drug Administration (FDA), the U.S. National Institutes of Health, and the stem cell research community have collaborated on a series of workshops that address moving pluripotent stem cell therapies into the clinic. The first two workshops in the series focused on preclinical science, and a third, future workshop will focus on clinical trials. This summary addresses major points from both of the recent preclinically focused meetings.
Recently, the U.S. Food and Drug Administration (FDA), the U.S. National Institutes of Health, and the stem cell research community have collaborated on a series of workshops that address moving pluripotent stem cell therapies into the clinic. The first two workshops in the series focused on preclinical science, and a third, future workshop will focus on clinical trials. This summary addresses major points from both of the recent preclinically focused meetings. When entering into a therapeutics developmental program based on pluripotent cells, investigators must make decisions at the very early stages that will have major ramifications during later phases of development. Presentations and discussions from both invited participants and FDA staff described the need to characterize and document the quality, variability, and suitability of the cells and commercial reagents used at every translational stage. This requires consideration of future regulatory requirements, ranging from donor eligibility of the original source material to the late-stage manufacturing protocols. Federal, industrial, and academic participants agreed that planning backward is the best way to anticipate what evidence will be needed to justify human testing of novel therapeutics and to eliminate wasted efforts.
Pluripotent stem cells; Stem cells; Clinical translation; Clinical trials
Morizane et al. (2013) show that donor-matched differentiated derivatives of induced pluripotent stem cells (iPSC) do not cause an immune response after transplantation, whereas transplantation of HLA-mismatched iPSC derivatives to the same site clearly does. The importance of these results is discussed in this commentary as we assess how best to move forward with iPSC-based cell therapy.
The US has had a very successful model for facilitating the translation of a basic discovery to a commercial application. The success of the model has hinged on providing clarity on ownership of a discovery, facilitating the licensing process, providing adequate incentive to the inventors, and developing a self-sustaining model for reinvestment. In recent years, technological, political, and regulatory changes have put strains on this model and in some cases have hindered progress rather than facilitated it. This is particularly true for the nascent field of regenerative medicine. To illustrate this, I will describe the contributing practices of several different entities, including universities, repositories, patent trolls, and service providers. It is my hope that the scientific community will be motivated to coordinate efforts against these obstacles to translation.
The remarkable speed with which the field of stem cell biology has evolved is unprecedented and has already changed the way we do science. In this series of articles we have invited leading experts to present their efforts in moving from the bench to the bedside, with the hope that we can learn from the experiences of the pioneers.
Human embryonic stem cell (hESC)-derived dopaminergic (DA) neurons hold potential for treating Parkinson’s disease (PD) through cell replacement therapy. Generation of DA neurons from hESCs has been achieved by co-culture with the stromal cell line PA6, a source of stromal cell-derived inducing activity (SDIA). However, the factor(s) produced by stromal cells that constitute SDIA are largely undefined. We previously reported that medium conditioned by PA6 cells can generate functional DA neurons from NTera2 human embryonal carcinoma stem cells. Here we show that PA6-conditioned medium can induce DA neuronal differentiation in both NTera2 cells and the hESC I6 cell line. To identify the factor(s) responsible for SDIA, we used large-scale microarray analysis of gene expression combined with mass spectrometric analysis of PA6-conditioned medium (CM). The candidate factors, hepatocyte growth factor (HGF), stromal cell-derived factor-1 α (SDF1α), secreted frizzled-related protein 1 (sFRP1), and vascular endothelial growth factor D (VEGFD) were identified and their concentrations in PA6 CM were established by immunoaffinity capillary electrophoresis. Upon addition of SDF1α, sFRP1 and VEGFD to the culture medium we observed an increase in the number of cells expressing tyrosine hydroxylase (a marker for DA neurons) and beta-III tubulin (a marker for immature neurons) in both the NTera2 and I6 cell lines. These results indicate that SDF1α, sFRP1 and VEGFD are major components of SDIA, and suggest the potential use of these defined factors to elicit DA differentiation of pluripotent human stem cells for therapeutic intervention in PD.
dopaminergic neurons; neuronal differentiation; stromal cell derived inducing activity; embryonic stem cells
Prospective donation of tissue specimens for induced pluripotent stem cell (iPSC) research requires an approach to informed consent that is constructed for this context. Approaches to informed consent have been variable in ways that threaten the simultaneous goals of protecting donors and safeguarding future research and translation, and investigators are seeking guidance. This analysis addresses this need by providing concrete recommendations for informed consent that balance the goals of iPSC and regenerative medicine researchers with the interests of individual research participants.
Induced pluripotent stem cells (iPSCs) have elicited excitement in both the scientific and ethics communities for their potential to advance basic and translational research. They have been hailed as an alternative to derivation from embryos that provides a virtually unlimited source of pluripotent stem cells for research and therapeutic applications. However, research with iPSCs is ethically complex, uniquely encompassing the concerns associated with genomics, immortalized cell lines, transplantation, human reproduction, and biobanking. Prospective donation of tissue specimens for iPSC research thus requires an approach to informed consent that is constructed for this context. Even in the nascent stages of this field, approaches to informed consent have been variable in ways that threaten the simultaneous goals of protecting donors and safeguarding future research and translation, and investigators are seeking guidance. We address this need by providing concrete recommendations for informed consent that balance the perspectives of a variety of stakeholders. Our work combines analysis of consent form language collected from investigators worldwide with a conceptual balancing of normative ethical concerns, policy precedents, and scientific realities. Our framework asks people to consent prospectively to a broad umbrella of foreseeable research, including future therapeutic applications, with recontact possible in limited circumstances. We argue that the long-term goals of regenerative medicine, interest in sharing iPSC lines, and uncertain landscape of future research all would be served by a framework of ongoing communication with donors. Our approach balances the goals of iPSC and regenerative medicine researchers with the interests of individual research participants.
Clinical translation; Ethics; iPS; Induced pluripotent stem cells
Recent advances in stem cell technology have enabled large scale production of human cells such as cardiomyocytes, hepatocytes and neurons for evaluation of pharmacological effect and toxicity of drug candidates. The assessment of compound efficacy and toxicity using human cells should lower the high clinical attrition rates of drug candidates by reducing the impact of species differences on drug efficacy and toxicity from animal studies. Methyl-β-cyclodextrin (MBCD) has shown to reduce lysosomal cholesterol accumulation in skin fibroblasts derived from patients with Niemann Pick type C disease and in the NPC1−/− mouse model. However, the compound has never been tested in human differentiated neurons. We have determined the cholesterol reduction effect of MBCD in neurons differentiated from human neural stem cells and commercially available astrocytes. The use of NSCs for producing differentiated neurons in large quantities can significantly reduce the production time and enhance the reproducibility of screening results. The EC50 values of MBCD on cholesterol reduction in human neurons and astrocytes were 66.9 and 110.7 µM, respectively. The results indicate that human neurons differentiated from the NSCs and human astrocytes are useful tools for evaluating pharmacological activity and toxicity of drug candidates to predict their clinical efficacy.
induced pluripotent stem cells; neural stem cells; human neurons; astrocytes; skin fibroblasts; methyl-β-cyclodextrin
The neural crest (NC) is a transient, multipotent, migratory cell population unique to vertebrates that gives rise to diverse cell lineages. Much of our knowledge of NC development comes from studies of organisms such as chicken and zebrafish because human NC is difficult to obtain because of its transient nature and the limited availability of human fetal cells. Here we examined the process of NC induction from human pluripotent stem cells, including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). We showed that NC cells could be efficiently induced from hESCs by a combination of growth factors in medium conditioned on stromal cells and that NC stem cells (NCSCs) could be purified by p75 using fluorescence-activated cell sorting (FACS). FACS-isolated NCSCs could be propagated in vitro in five passages and cryopreserved while maintaining NCSC identity characterized by the expression of a panel of NC markers such as p75, Sox9, Sox10, CD44, and HNK1. In vitro-expanded NCSCs were able to differentiate into neurons and glia (Schwann cells) of the peripheral nervous system, as well as mesenchymal derivatives. hESC-derived NCSCs appeared to behave similarly to endogenous embryonic NC cells when injected in chicken embryos. Using a defined medium, we were able to generate and propagate a nearly pure population of Schwann cells that uniformly expressed glial fibrillary acidic protein, S100, and p75. Schwann cells generated by our protocol myelinated rat dorsal root ganglia neurons in vitro. To our knowledge, this is the first report on myelination by hESC- or iPSC-derived Schwann cells.
Neural crest; Human embryonic stem cells; Differentiation; Schwann cells; Peripheral neuron
Schwann cells (SCs) play an important role in the pathogenesis of peripheral nerve diseases and represent a potential target for development of therapies. However, use of primary human SCs (hSCs) for in vitro models is limited because these cells are difficult to prepare and maintain in high yield and purity under common cell culture conditions. To circumvent this obstacle, we immortalized primary human fetal SCs using the SV40 large T-antigen and human telomerase reverse transcriptase expression vectors. After cloning, selection, and purification, we evaluated several immortalized SC lines for their ability to express extracellular matrix (ECM) molecules and myelinate embryonic rat sensory axons. In addition, we established a gene expression profile and explored their sensitivity to oxidative stress in a simple in vitro assay. Immortalized hSC clones expressed common glial markers and a broad variety of growth factors, receptors, and ECM molecules as determined by immunocytochemistry, microarray, and quantitative reverse transcription–polymerase chain reaction. In neuron-SC co-cultures, these cells were able to myelinate rat dorsal root ganglia neurons, although their effectiveness was lower in comparison to primary rat SCs. In toxicity assays, immortalized hSCs remain susceptible to oxidative stress induced by H2O2. This study shows that, using specific immortalization techniques, it is possible to establish hSC lines that retain characteristics of typical primary hSCs. These cells are particularly useful for drug screening and studies aimed at disease mechanisms involving SCs.
Although astrocytes are involved in the production of an inhibitory glial scar following injury, they are also capable of providing neuroprotection and supporting axonal growth. There is growing appreciation for a diverse and dynamic population of astrocytes, specified by a variety of glial precursors, whose function is regulated regionally and temporally. Consequently, the therapeutic application of glial precursors and astrocytes by effective transplantation protocols requires a better understanding of their phenotypic and functional properties and effective protocols for their preparation. We present a systematic analysis of astrocyte differentiation using multiple preparations of glial-restricted precursors (GRP), evaluating their morphological and phenotypic properties following treatment with fetal bovine serum (FBS), bone morphogenetic protein 4 (BMP-4), or ciliary neurotrophic factor (CNTF) in comparison to controls treated with basic fibroblast growth factor (bFGF), which maintains undifferentiated GRP. We found that treatments with FBS or BMP-4 generated similar profiles of highly differentiated astrocytes that were A2B5−/GFAP+. Treatment with FBS generated the most mature astrocytes, with a distinct and nearhomogeneous morphology of fibroblast-like flat cells, whereas BMP-4 derived astrocytes had a stellate, but heterogeneous morphology. Treatment with CNTF induced differentiation of GRP to an intermediate state of GFAP+ cells that maintained immature markers and had relatively long processes. Furthermore, astrocytes generated by BMP-4 or CNTF showed considerable experimental plasticity, and their morphology and phenotypes could be reversed with complementary treatments along a wide range of mature-immature states. Importantly, when GRP or GRP treated with BMP-4 or CNTF were transplanted acutely into a dorsal column lesion of the spinal cord, cells from all 3 groups survived and generated permissive astrocytes that supported axon growth and regeneration of host sensory axons into, but not out of the lesion. Our study underscores the dynamic nature of astrocytes prepared from GRP and their permissive properties, and suggest that future therapeutic applications in restoring connectivity following CNS injury are likely to require a combination of treatments.
bone morphogenetic protein (BMP); ciliary neurotrophic factor (CNTF); astrocyte differentiation; astrocyte transplantation; spinal cord injury; axon regeneration
The discovery of the ability to induce somatic cells to a pluripotent state through the overexpression of specific transcription factors has the potential to transform the ways in which pharmaceutical agents and cellular transplantation therapies are developed. Proper utilization of the technology to generate induced pluripotent stem cells (iPSCs) requires that researchers select the appropriate reprogramming method for generating iPSCs so that the resulting iPSCs can be transitioned towards clinical applications effectively. This article reviews all of the currently available reprogramming techniques with a focus on critiquing them on the basis of their utility in translational medicine.
INDUCED PLURIPOTENT STEM CELLS; REPROGRAMMING METHODS; TRANSLATIONAL MEDICINE