Glycans define the stemness of naïve and primed pluripotent stem cells

Cell surface glycans are tissue-specific and developmentally regulated. They function as essential modulators in cell-cell interactions, cell-extracellular matrix interactions, and ligand-receptor interactions, binding to various ligands, including Wnt, fibroblast growth factors, and bone morphogenetic proteins. Embryonic stem (ES) cells, originally derived from the inner cell mass of blastocysts, have the essential characteristics of pluripotency and self-renewal. Recently, it has been proposed that mouse and human conventional ES cells are present in different developmental stages, namely pre-implantation blastocyst and post-implantation blastocyst stages, also called the naïve state and the primed state, respectively. They therefore require different extrinsic signals for the maintenance of self-renewal and pluripotency, and also appear to require different surface glycans. Understanding of molecular mechanisms involving glycans in self-renewal and pluripotency of ES cells is increasingly important for potential clinical applications, as well as for basic research. This review focuses on the roles of glycans in the two different states of pluripotent stem cells, namely the naïve state and the primed state, and the transition between these two states.     

Porcine induced pluripotent stem cells analogous to naïve and primed embryonic stem cells of the mouse

Authentic or na?ve embryonic stem cells (ESC) have probably never been derived from the inner cell mass (ICM) of pig blastocysts, despite over 25 years of effort. Recently, several groups, including ours, have reported induced pluripotent stem cells (iPSC) from swine by reprogramming somatic cells with a combination of four factors, OCT4 (POU5F1)/SOX2/KLF4/c-MYC delivered by retroviral transduction. The porcine (p) iPSC resembled human (h) ESC and the mouse "Epiblast stem cells" (EpiSC) in their colony morphology and expression of pluripotent genes, and are likely dependent on FGF2/ACTIVIN/NODAL signaling, therefore representing a primed ESC state. These cells are likely to advance swine as a model in biomedical research, since grafts could potentially be matched to the animal that donated the cells for re-programming. The objective of the present work has been to develop na?ve piPSC. Employing a combination of seven reprogramming factors assembled on episomal vectors, we successfully reprogrammed porcine embryonic fibroblasts on a modified LIF-medium supplemented with two kinase inhibitors; CHIR99021, which inhibits GSK-3beta, and PD0325901, a MEK inhibitor. The derived piPSC bear a striking resemblance to na?ve mESC in colony morphology, are dependent on LIF to maintain an undifferentiated phenotype, and express markers consistent with pluripotency. They exhibit high telomerase activity, a short cell cycle interval, and a normal karyotype, and are able to generate teratomas. Currently, the competence of these lines for contributing to germ-line chimeras is being tested.     

Search for nave human pluripotent stem cells

Normal mouse pluripotent stem cells were originally derived from the inner cell mass(ICM) of blastocysts and shown to be the in vitro equivalent of those pre-implantation embryonic cells, and thus were called embryonic stem cells(ESCs). More than a decade later, pluripotent cells were isolated from the ICM of human blastocysts. Despite being called human ESCs, these cells differ significantly from mouse ESCs, including different morphology and mechanisms of control of pluripotency, suggesting distinct embryonic origins of ESCs from the two species. Subsequently, mouse pluripotent stem cells were established from the ICMderived epiblast of post-implantation embryos. These mouse epiblast stem cells(Epi SCs) are morphological and epigenetically more similar to human ESCs. This raised the question of whether cells from the human ICM are in a more advanced differentiation stage than their murine counterpart, or whether the available culture conditions were not adequate to maintain those human cells in their in vivo state, leading to a transition into Epi SC-like cells in vitro. More recently, novel culture conditions allowed the conversion of human ESCs into mouse ESC-like cells called nave(or ground state) human ESCs, and the derivation of nave human ESCs from blastocysts. Here we will review the characteristics of each type of pluripotent stem cells, how(and whether) these relate to different stages of embryonic development, and discuss the potential implications of nave human ESCs in research and therapy.     

Characterization and genetic manipulation of primed stem cells into a functional na?ve state with ESRRB

AIM To identify differences between primed mouse embryonic stem cells(ESCs) and fully functional naive ESCs; to manipulate primed cells into a naive state. METHODS We have cultured 3 lines of cells from different mouse strains that have been shown to be naive or primed as determined by generating germline-transmitting chimeras.Cells were put through a battery of tests to measure the different features. RNA from cells was analyzed using microarrays, to determine a priority list of the differentially expressed genes. These were later validated by quantificational real-time polymerase chain reaction. Viral cassettes were created to induce expression of differentially expressed genes in the primed cells through lentiviral transduction. Primed reprogrammed cells were subjected to in-vivo incorporation studies.RESULTS Most results show that both primed and naive cells have similar features(morphology, proliferation rates, stem cell genes expressed). However, there were some genes that were differentially expressed in the na?ve cells relative to the primed cells. Key upregulated genes in na?ve cells include ESRRB, ERAS, ATRX, RNF17, KLF-5, and MYC. After over-expressing some of these genes the primed cells were able to incorporate into embryos in-vivo, re-acquiring a feature previously absent in these cells. CONCLUSION Although there are no notable phenotypic differences, there are key differences in gene expression between these na?ve and primed stem cells. These differences can be overcome through overexpression.     

Different roles of TGF-β in the multi-lineage differentiation of stem cells

Stem cells are a population of cells that has infinite or long-term self-renewal ability and can produce various kinds of descendent cells.Transforming growth factor β(TGF-β) family is a superfamily of growth factors,including TGF-β1,TGF-β2 and TGF-β3,bone morphogenetic proteins,activin/inhibin,and some other cytokines such as nodal,which plays very important roles in regulating a wide variety of biological processes,such as cell growth,differentiation,cell death.TGF-β,a pleiotropic cytokine,has been proved to be differentially involved in the regulation of multi-lineage differentiation of stem cells,through the Smad pathway,non-Smad pathways including mitogen-activated protein kinase pathways,phosphatidylinositol-3-kinase/AKT pathways and Rholike GTPase signaling pathways,and their cross-talks.For instance,it is generally known that TGF-β promotes the differentiation of stem cells into smooth muscle cells,immature cardiomyocytes,chondrocytes,neurocytes,hepatic stellate cells,Th17 cells,and dendritic cells.However,TGF-β inhibits the differentiation of stem cells into myotubes,adipocytes,endothelial cells,and natural killer cells.Additionally,TGF-β can provide competence for early stages of osteoblastic differentiation,but at late stages TGF-β acts as an inhibitor.The three mammalian isoforms(TGF-β1,2 and 3) have distinct but overlapping effects on hematopoiesis.Understanding the mechanisms underlying the regulatory effect of TGF-β in the stem cell multi-lineage differentiation is of importance in stem cell biology,and will facilitate both basic research and clinical applications of stem cells.In this article,we discuss the current status and progress in our understanding of different mechanisms by which TGF-β controls multi-lineage differentiation of stem cells.     

The transition of mouse pluripotent stem cells from the naïve to the primed state requires Fas signaling through 3-O sulfated heparan sulfate structures recognized by the HS4C3 antibody

The characteristics of pluripotent embryonic stem cells of human and mouse are different. The properties of human embryonic stem cells (hESCs) are similar to those of mouse epiblast stem cells (mEpiSCs), which are in a later developmental pluripotency state, the so-called “primed state” compared to mouse embryonic stem cells (mESCs) which are in a naïve state. As a result of the properties of the primed state, hESCs proliferate slowly, cannot survive as single cells, and can only be transfected with genes at low efficiency. Generating hESCs in the naïve state is necessary to overcome these problems and allow their application in regenerative medicine. Therefore, clarifying the mechanism of the transition between the naïve and primed states in pluripotent stem cells is important for the establishment of stable methods of generating naïve state hESCs. However, the signaling pathways which contribute to the transition between the naïve and primed states are still unclear. In this study, we carried out induction from mESCs to mEpiSC-like cells (mEpiSCLCs), and observed an increase in the activation of Fas signaling during the induction. The expression of Fgf5, an epiblast marker, was diminished by inhibition of Fas signaling using the caspase-8 and -3 blocking peptides, IETD and DEVD, respectively. Furthermore, during the induction, we observed increased expression of 3-O sulfated heparan sulfate (HS) structures synthesized by HS 3-O-sulfotransferase (3OST), which are recognized by the HS4C3 antibody (HS4C3-binding epitope). Knockdown of 3OST-5 reduced Fas signaling and the potential for the transition to mEpiSCLCs. This indicates that the HS4C3-binding epitope is necessary for the transition to the primed state. We propose that Fas signaling through the HS4C3-binding epitope contributes to the transition from the naïve state to the primed state.     

Long-term self-renewal of naïve neural stem cells in a defined condition

During embryonic development, neural stem cells (NSCs) emerge as early as the neural plate stage and give rise to the nervous system. Early-stage NSCs express Sry-related-HMG box-1 (Sox1) and are biased towards neuronal differentiation. However, long-term maintenance of early-stage NSCs in vitro remains a challenge. Here, we report development of a defined culture condition for the long-term maintenance of Sox1-positive early-stage mouse NSCs. The proliferative ability of these Sox1-positive NSCs was confirmed by clonal propagation. Compared to the NSCs cultured using the traditional culture condition, the long-term self-renewing Sox1-positive NSCs efficiently differentiate into neurons and exhibit an identity representative of the anterior and midbrain regions. These early-stage Sox1-positive NSCs could also be switched to late-stage NSCs by being cultured with bFGF/EGF, which can then differentiate into astrocytes and oligodendrocytes. The long-term self-renewing Sox1-positive NSCs were defined as naïve NSCs, based on their high neuronal differentiation capacity and anterior regional identity. This culture condition provides a robust platform for further dissection of the NSC self-renewal mechanism and promotes potential applications of NSCs for cell-based therapy on nervous system disorders.     

Short-term serum-free culture reveals that inhibition of Gsk3β induces the tumor-like growth of mouse embryonic stem cells

Here, we present evidence that the tumor-like growth of mouse embryonic stem cells (mESCs) is suppressed by short-term serum-free culture, which is reversed by pharmacological inhibition of Gsk3β. Mouse ESCs maintained under standard conditions using fetal bovine serum (FBS) were cultured in a uniquely formulated chemically-defined serum-free (CDSF) medium, namely ESF7, for three passages before being subcutaneously transplanted into immunocompromised mice. Surprisingly, the mESCs failed to produce teratomas for up to six months, whereas mESCs maintained under standard conditions generated well-developed teratomas in five weeks. Mouse ESCs cultured under CDSF conditions maintained the expression of Oct3/4, Nanog, Sox2 and SSEA1, and differentiated into germ cells in vivo. In addition, when mESCs were cultured under CDSF conditions supplemented with FBS, or when the cells were cultured under CDSF conditions followed by standard culture conditions, they consistently developed into teratomas. Thus, these results validate that the pluripotency of mESCs was not compromised by CDSF conditions. Mouse ESCs cultured under CDSF conditions proliferated significantly more slowly than mESCs cultured under standard conditions, and were reminiscent of Eras-null mESCs. In fact, their slower proliferation was accompanied by the downregulation of Eras and c-Myc, which regulate the tumor-like growth of mESCs. Remarkably, when mESCs were cultured under CDSF conditions supplemented with a pharmacological inhibitor of Gsk3β, they efficiently proliferated and developed into teratomas without upregulation of Eras and c-Myc, whereas mESCs cultured under standard conditions expressed Eras and c-Myc. Although the role of Gsk3β in the self-renewal of ESCs has been established, it is suggested with these data that Gsk3β governs the tumor-like growth of mESCs by means of a mechanism different from the one to support the pluripotency of ESCs.     

Differential expression of FCRLA in naïve and activated mouse B cells

FCRLA is an intracellular B cell protein that belongs to the FcR-like family. Using newly generated FCRLA-specific antibodies, we studied the constitutive expression pattern of mouse FCRLA and monitored changes during an immune response and following in vitro B cell activation. All B cell subpopulations examined expressed FCRLA. However, the level of FCRLA expression is determined by the stage of B cell differentiation. Low expression of FCRLA is characteristic of naïve follicular and marginal zone B cells. High expression was detected in a small fraction of activated B cells scattered along migratory pathways in the lymphoid tissues. FCRLA-bright cells could be subdivided into two subpopulations, with high and low/undetectable level of intracellular immunoglobulins, which phenotypically resemble either plasma or memory B cells. High expression of FCRLA in subset(s) of terminally differentiated B-cells suggests that, being an ER protein, FCRLA may participate in the regulation of immunoglobulin assembly and secretion.     

Non-random tissue distribution of human naïve umbilical cord matrix stem cells

AIM: To determine the tissue and temporal distribution of human umbilical cord matrix stem (hUCMS) cells in severe combined immunodeficiency (SCID) mice. METHODS: For studying the localization of hUCMS cells, tritiated thymidine-labeled hUCMS cells were injected in SCID mice and tissue distribution was quantitatively determined using a liquid scintillation counter at days 1, 3, 7 and 14. Furthermore, an immunofluorescence detection technique was employed in which anti-human mitochondrial antibody was used to identify hUCMS cells in mouse tissues. In order to visualize the distribution of transplanted hUCMS cells in H&E stained tissue sections, India Black ink 4415 was used to label the hUCMS cells. RESULTS: When tritiated thymidine-labeled hUCMS cells were injected systemically (iv) in female SCID mice, the lung was the major site of accumulation at 24 h after transplantation. With time, the cells migrated to other tissues, and on day three, the spleen, stomach, and small and large intestines were the major accumulation sites. On day seven, a relatively large amount of radioactivity was detected in the adrenal gland, uterus, spleen, lung, and digestive tract. In addition, labeled cells had crossed the blood brain barrier by day 1. CONCLUSION: These results indicate that peripherally injected hUCMS cells distribute quantitatively in a tissue-specific manner throughout the body.     

Different genome stability proteins underpin primed and na?ve adaptation in E. coli CRISPR-Cas immunity

CRISPR-Cas is a prokaryotic immune system built from capture and integration of invader DNA into CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci, termed ‘Adaptation’, which is dependent on Cas1 and Cas2 proteins. In Escherichia coli, Cascade-Cas3 degrades invader DNA to effect immunity, termed ‘Interference’. Adaptation can interact with interference (‘primed’), or is independent of it (‘naïve’). We demonstrate that primed adaptation requires the RecG helicase and PriA protein to be present. Genetic analysis of mutant phenotypes suggests that RecG is needed to dissipate R-loops at blocked replication forks. Additionally, we identify that DNA polymerase I is important for both primed and naive adaptation, and that RecB is needed for naïve adaptation. Purified Cas1-Cas2 protein shows specificity for binding to and nicking forked DNA within single strand gaps, and collapsing forks into DNA duplexes. The data suggest that different genome stability systems interact with primed or naïve adaptation when responding to blocked or collapsed invader DNA replication. In this model, RecG and Cas3 proteins respond to invader DNA replication forks that are blocked by Cascade interference, enabling DNA capture. RecBCD targets DNA ends at collapsed forks, enabling DNA capture without interference. DNA polymerase I is proposed to fill DNA gaps during spacer integration.     

Expression of TGFβ family factors and FGF2 in mouse and human embryonic stem cells maintained in different culture systems

Mouse and human embryonic stem cells are in different states of pluripotency (naive/ground and primed states). Mechanisms of signaling regulation in cells with ground and primed states of pluripotency are considerably different. In order to understand the contribution of endogenous and exogenous factors in the maintenance of a metastable state of the cells in different phases of pluripotency, we examined the expression of TGFβ family factors (ActivinA, Nodal, Lefty1, TGFβ1, GDF3, BMP4) and FGF2 initiating the appropriate signaling pathways in mouse and human embryonic stem cells (mESCs, hESCs) and supporting feeder cells. Quantitative real-time PCR analysis of gene expression showed that the expression patterns of endogenous factors studied were considerably different in mESCs and hESCs. The most significant differences were found in the levels of endogenous expression of TGFβ1, BMP4 and ActivinA. The sources of exogenous factors ActivnA, TGFβ1, and FGF2 for hESCs are feeder cells (mouse and human embryonic fibroblasts) expressing high levels of these factors, as well as low levels of BMP4. Thus, our data demonstrated that the in vitro maintenance of metastable state of undifferentiated pluripotent cells is achieved in mESCs and hESCs using different schemes of the regulations of ActivinA/Nodal/Lefty/Smad2/3 and BMP/Smad1/5/8 endogenous branches of TGFβ signaling. The requirement for exogenous stimulation or inhibition of these signaling pathways is due to different patterns of endogenous expression of TGFβ family factors and FGF2 in the mESCs and hESCs. For the hESCs, enhanced activity of ActivinA/Nodal/Lefty/Smad2/3 signaling by exogenous factor stimulation is necessary to mitigate the effects of BMP/Smad1/5/8 signaling pathways that promote cell differentiation into the extraembryonic structures. Significant differences in endogenous FGF2 expression in the cells in the ground and primed states of pluripotency demonstrate diverse involvement of this factor in the regulation of the pluripotent cell self-renewal.     

Stage-specific signaling through TGFβ family members and WNT regulates patterning and pancreatic specification of human pluripotent stem cells

The generation of insulin-producing β-cells from human pluripotent stem cells is dependent on efficient endoderm induction and appropriate patterning and specification of this germ layer to a pancreatic fate. In this study, we elucidated the temporal requirements for TGFβ family members and canonical WNT signaling at these developmental stages and show that the duration of nodal/activin A signaling plays a pivotal role in establishing an appropriate definitive endoderm population for specification to the pancreatic lineage. WNT signaling was found to induce a posterior endoderm fate and at optimal concentrations enhanced the development of pancreatic lineage cells. Inhibition of the BMP signaling pathway at specific stages was essential for the generation of insulin-expressing cells and the extent of BMP inhibition required varied widely among the cell lines tested. Optimal stage-specific manipulation of these pathways resulted in a striking 250-fold increase in the levels of insulin expression and yielded populations containing up to 25% C-peptide+ cells.     

New frontiers in human cell biology and medicine: Can pluripotent stem cells deliver?

Human pluripotent stem cells provide enormous opportunities to treat disease using cell therapy. But human stem cells can also drive biomedical and cell biological discoveries in a human model system, which can be directly linked to understanding disease or developing new therapies. Finally, rigorous scientific studies of these cells can and should inform the many science and medical policy issues that confront the translation of these technologies to medicine. In this paper, I discuss these issues using amyotrophic lateral sclerosis as an example.Much of modern cell biological discovery has been driven by the study of a diverse variety of primary and transformed cultured cells. However, the advent of human pluripotent cells is providing new avenues of discovery. These cells are genetically manipulable, euploid, expandable to large numbers, and can differentiate to most if not all human cell types. In addition, these cells can be used to analyze the large number of mutations and diverse genetic variation present in large human populations. In fact, not only are human pluripotent stem cells useful for typical cell culture experiments, but they are amenable to many of the types of genetic and molecular genetic approaches that historically have only been feasible in genetic and developmental systems, such as Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans, or mouse.There are two types of human pluripotent stem cells in use. Human embryonic stem cells (hESC) are derived from human embryos that would otherwise be discarded and that are generally donated with substantial informed consent and ethical requirements (Shamblott et al., 1998; Thomson et al., 1998). Human induced pluripotent stem cells (hIPSC) are generated by several different reprogramming technologies, generally from fibroblasts obtained from small skin biopsies or other human somatic cell types, such as blood (Takahashi et al., 2007). Recent work suggests that hESC and hIPSC, although not identical in their properties, share very important features. First, hESC and hIPSC are both pluripotent so that any cell type of interest can in principle be generated. In fact, differentiation methods for many types of specialized human cells are being developed rapidly, fueled in large part by the need to generate stable differentiated derivatives for cell therapy. Second, hIPSC and hESC can be handled in the laboratory under conditions that are relatively straightforward for skilled cell culture scientists. Third, both hIPSC and hESC, when handled properly, are genetically relatively stable with a diploid karyotype so that gene dose and gene expression at all loci is effectively “normal” or at least representative of expression levels in cells in the intact human. These properties make these cells or their differentiated derivatives suitable for genetic screens using RNA interference, small molecules, insertional mutagenesis, or other analogous tools. Important differences between hESC and hIPSC include an apparent elevation in mutation load in hIPSC (Gore et al., 2011) and differences in epigenetic state. Either of these features may substantially influence the behavior of each cell type and its differentiated derivatives. Thus, hESC and hIPSC may play different roles in the discovery of new cellular and disease mechanisms and in the development of cellular therapies. Recent examples of novel discoveries made using hESC and hIPSC include substantial new insights into the control of the pluripotent state itself and identification of molecular pathways controlling cellular differentiation.

Disease in a dish approaches with hESC and hIPSC

Although hESC and hIPSC are just beginning to be used to probe and elucidate new cellular processes, there is already substantial progress using pluripotent stem cell approaches to unravel disease mechanisms using so-called disease in a dish paradigms (Unternaehrer and Daley, 2011). Disease in a dish methods use gene manipulation and/or reprogramming technologies to generate hESC or hIPSC lines with genomes carrying known mutations causing human disease, lesions such as shRNA expression mimicking human disease mutations (Marchetto et al., 2010; Ordonez et al., 2012), or genomes carrying combinations of known or unknown variants that contribute to disease (Fig. 1). With the advent of powerful molecular tools, such as Tal effector nucleases, individual genes can be manipulated by introducing point mutations with great precision (Hockemeyer et al., 2011). Thus, disease-causing mutations or other genetic lesions can be studied for their impacts on cellular processes under “normal” conditions of gene expression and in different genetic backgrounds. Similarly, suppressor and enhancer studies are feasible and will help unravel poorly understood cellular mechanisms. Finally, after differentiation to specialized cell types, cellular mechanisms and interactions as well as disease and potential therapies can be evaluated in bona fide human cells. These approaches are in their infancy but have substantial potential given the limitations of mouse models of disease to accurately recapitulate human disease and the many obvious differences between the details of mouse physiology and human physiology. They also bring unique advantages for diseases in which the key cell types, e.g., human central nervous system neurons, are difficult if not impossible to obtain in good condition or early in disease.Open in a separate windowFigure 1.Disease in a dish. Stem cells can be used to analyze how human genetic variation or mutation contributes to defined cellular phenotypes. Using neuronal phenotype as an example, Tal effector nucleases (TALENs) can be used to make defined changes in hIPSC of a common genetic background to analyze the contribution of a defined mutation or more complex variation to neuronal phenotypes.Examples of recent disease in dish studies include a variety of neurodegenerative diseases, heart diseases, and diseases of other organ systems (Unternaehrer and Daley, 2011). Although this work is in its early stages, there is a great deal of potential for meaningful mechanistic cell biological research in this collection of intriguing areas. In addition, these disease in dish models provide unique human materials for direct testing of drug safety and efficacy.

Amyotrophic lateral sclerosis (ALS): A disease in a dish paradigm

ALS, also known as Lou Gehrig’s disease, provides an intriguing example that illustrates the path from mechanism-based research to understanding and potential therapies using disease in a dish approaches. ALS is a disease in which death of motor neurons causes paralysis of voluntary muscles. As the disease progresses, paralysis ultimately extends to the muscles involved in breathing, swallowing, and all other voluntary movements. Once diagnosed, ALS generally causes death within 3–5 yr or less. There is only one approved drug for ALS, riluzole, but individual patients do not perceive much benefit because riluzole generates statistical prolongation of life for only a few months based on large-scale clinical trials. The key problem of course is to learn what causes death of motor neurons in ALS and whether this information might help develop a therapy that protects motor neurons from dysfunction and death.Although most ALS is “sporadic,” some forms are hereditary, including a form caused by mutations in the gene encoding dismutase SOD1 (superoxide 1). The generation of transgenic mouse and rat models that carry human mutant SOD1 genes has led to substantial progress in the understanding of cellular mechanisms that contribute to disease. In particular, a series of genetic studies in transgenic and chimeric mice led to the realization that the death of motor neurons in ALS might not be cell autonomous (Clement et al., 2003; Boillée et al., 2006; Yamanaka et al., 2008a,b). Disease in a dish studies using astrocytes carrying SOD1 mutant genes mixed with in vitro differentiated motor neurons made from pluripotent stem cells confirmed these findings and lead directly to searches for secreted toxic factors and drug testing (Di Giorgio et al., 2007, 2008; Marchetto et al., 2008). The general conclusion from these studies is that motor neuron death in ALS is strongly influenced by other cells in the spinal cord that make important contributions to, or protect from, motor neuron death. Whether astrocytes, microglia, or other cell types carry mutant SOD1 genes determines whether they exhibit stimulatory or protective effects on motor neuron death. Although such conclusions might be limited to SOD1-mediated ALS, there is also recent evidence that astrocytes might contribute to sporadic ALS as well (Haidet-Phillips et al., 2011).

Development of cell replacement or augmentation therapies

Successfully treating debilitating and currently incurable diseases with cell replacement or augmentation therapies requires basic cell biological research to fuel the generation and testing of new therapies (Fig. 2). For example, expansion of hematopoietic stem cell therapeutic approaches from leukemias to other diseases is based on a sound understanding of the basic cell and developmental biology of these cells. Several different stem cell therapies are in the midst of development and testing for several disorders, including spinal cord injury, graft versus host disease, skin diseases, blindness, diabetes, and AIDS (e.g., California Institute for Regenerative Medicine, 2009; Pollack, 2012; Schwartz et al., 2012). Using ALS as an example, some stem cell–based therapy efforts are aimed at trying to replace motor neurons that are damaged or die in ALS. But inducing motor neurons or their stem cell precursors to engraft into spinal cords or upper motor cortex and then appropriately extend axons and wire to peripheral muscles may be a challenge that will take many years to solve. Interestingly, the evidence that ALS is non–cell autonomous with major contributions from astrocytes and other glia has led to two different categories of cell therapy approach. One approach, which I regard as little more than guesswork, has tried to treat ALS by introducing poorly defined mesenchymal stem cells or cord blood stem cells directly into the spinal cord of ALS model animals. In fact, even in the absence of strong and reliable evidence, a clinical trial of cord blood stem cells transplanted into the spinal cord of human ALS patients was launched by a private company (http://www.tcacellulartherapy.com/fda_clinical_trials.html) and then halted by the FDA. A more rational approach given the state of scientific understanding, the state of experiments in animal models, and the in vitro data is to introduce progenitor cells that can differentiate to astrocytes or progenitors that secrete growth factors (Klein et al., 2005; Suzuki et al., 2007; Lepore et al., 2008; Suzuki and Svendsen, 2008; Hefferan et al., 2012). One of these approaches has recently reached clinical trials using fetal-derived spinal cord stem cells in which one hopes that enough will be learned to support more trials using different stem cell–generated preparations and perhaps different surgical methods or spinal cord sites.Open in a separate windowFigure 2.Cell replacement therapy using stem cells. A possible path from patients with hereditary ALS to cell biological research using transgenic mouse or pluripotent stem cell models to cell therapies. In this example, assessing the contribution of different cell types to ALS through rigorous research can lead to a rational approach to cell therapy.

Driving evidence-based scientific and medical policy with stem cell–driven discovery

The social and medical issues that arise in the development of cell therapies are and will be heavily influenced by the scientific discoveries about and using human stem cells. These social and medical challenges are well illustrated by a discussion of ALS owing to its rapidly progressive and devastating nature.First is whether one type of therapy can treat all types of ALS patients. Solving this issue will require a better understanding of what causes ALS, what cellular mechanisms contribute to motor neuron death, and which cells contribute in different forms of ALS. One key and possibly false assumption that drives current efforts is that all forms of ALS will exhibit the type of cellular nonautonomy found in animal models of SOD1-mediated ALS. Thus, models of sporadic ALS and hereditary forms of ALS such as those mediated by FUS/TLS or TDP-43 mutations must be tested. These experiments will also allow tests of the magnitude of the relative contributions of different cell types to motor neuron death or rescue in different forms of ALS. Additionally, if the actual cellular pathways that are defective in astrocytes and motor neurons can be better defined, cellular augmentation strategies and drug discovery could take advantage of that information.Second is the so-called snake oil problem (http://www.closerlookatstemcells.org; CBSNews, 2010). Numerous misleading and probably fraudulent advertisements can be found about clinics offering stem cell cures for ALS. These wild claims ignore large amounts of scientific data about the nature of ALS and rational approaches to therapy and prey upon those who don’t have ready access to or cannot evaluate legitimate scientific and medical information. Our legitimate scientific and medical community needs to stand against these frauds and provide accurate information derived from rigorous research to patients so that they are not taken advantage of by these clinics. In addition, we must work to ensure that legitimate efforts are not damaged by the blowback from those who effectively steal from desperately ill patients and their families or the likely harm to these patients that is coming from clinics that dispense untested and sometimes dangerous therapies.Third is the cell tracking problem. Currently, it is difficult to know how cells transplanted into the spinal cord of an ALS patient behave until after a patient has died. In addition, using antibodies to examine postmortem material from a transplant patient is problematic because the transplants are of human cells into a human patient. We desperately need to develop safe and sensitive methods for cell marking and imaging that will allow us to track cell behavior in patients in real time after transplant. Real-time measures would allow therapy to be modified or even repeated based on the analysis of cell behavior. These methods will rely heavily on cell biological research to identify cellular pathways and markers that could be visualized in real time by magnetic resonance imaging, positron emission tomography, or other imaging modalities.Fourth is how to manage individual patient response versus the average response of patients in a clinical trial. Although most often thought about with respect to drug therapies, different forms of ALS might vary in their response to cell therapies. An interesting possibility is that hIPSC lines from individual patients could be used not only for drug testing but also for evaluating the genetically driven contribution of different cell types to each patient’s version of ALS. A corollary is that for ALS patients included in a clinical trial, the notion that cells would be introduced only once and that the patient would then be “passively” followed with no change in treatment paradigm until death might be unacceptable. In conventional medicine, one might try treatment again or modify treatment course, depending on how an individual patient responds. On the other hand, prospective design of a statistically rigorous clinical trial requires development of a treatment plan and identification of rigorous outcome measures that should not be modified if the data are to be interpretable. Development of new statistical methods, outcome measures, hIPSC evaluation of phenotypes, and perhaps, cellular marking methods might allow trials to tolerate modification as part of an ALS patient’s clinical care. Perhaps rigorous data from hIPSC-based research could be used to make a case to the FDA that ALS clinical trials need to be more responsive to patient needs and variable outcomes with a disease that is as complicated and clinically inconsistent as ALS.Fifth, and finally, is the risk benefit analysis that can hinder or accelerate the development of therapies for rapidly fatal diseases such as ALS. Current paradigms of therapy development are risk averse and require enormous amounts of information on safety and possible efficacy before trials can be approved, financed, and launched. Yet, some patient populations, such as those with ALS, when facing a near certain death sentence, are very risk tolerant and might be willing to participate in trials with much lower certainty. Our community must work with the FDA to tackle this problem and to perhaps dramatically accelerate the introduction of good, but perhaps radical, ideas that might work but in which safety or efficacy testing in animals could take many years, or simply be unreliable, so that current patients would have no hope of benefiting. I often ask myself, as I work with my colleagues to develop a cell-based therapy for ALS that has been partially tested in animals but is not yet complete and therefore not ready for humans, what I would do if I, my wife, or one of my children developed ALS. Would I be willing to have appropriate types of stem cells or their derivatives transplanted into them even if I were not absolutely certain and had not yet proven absolute safety or efficacy? Interestingly, I find that in thinking about this issue, I fall back on my scientific understanding of ALS and the rigorous types of data on ALS mechanisms in the mainstream scientific literature. The result is that my own risk tolerance rises substantially when I have the ability to consider published and unpublished data and how it might be applied. I think that all ALS patients should have this information and that the FDA should be responsive to these patients when they want to take well-informed risks with experimental therapies that may not yet meet current FDA standards. Clearly, the devil will be in the details for implementing such an approach and ensuring patient protection as well as opportunity, but we owe this consideration to current ALS patients and those with comparably severe diseases. Again, however, this is a debate in which rigorous scientific research can drive the agenda and resulting policies.

Concluding remarks

Virchow developed the concept that disease arises in the individual cells of a tissue (Schultz, 2008). This important principle is the foundation for using human stem cells to understand basic cellular mechanisms and to extend that understanding to the development of therapies. Treating disease by targeting the misbehaving cells is clearly a wonderful opportunity for therapy development and research. Thus, probing the secrets of human cells by taking advantage of human pluripotent stem cells may signal the dawn of a new era in cell biology research.Finally, consider the many remarkable discoveries and novel mechanisms found when the basic tools of cell and molecular biology were applied to unusual members of the model organism toolbox, including snakes, ciliates, planaria, jerboa, and other organisms that have developed unusual biological strategies during evolution. Could humans be added to this list, and could the study of basic human cell biology yield comparable discoveries? Because humans are a large, long-lived organism with a complex brain, a rich evolutionary history, and substantial genetic variation across large and accessible populations, the answer is certain to be yes.     

Generation of virus-free induced pluripotent stem cell clones on a synthetic matrix via a single cell subcloning in the naïve state

CD34+ cord blood cells can be reprogrammed effectively on dishes coated with a synthetic RGD motif polymer (PronectinF?) using a temperature sensitive Sendai virus vector (SeV TS7) carrying reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC. Dish-shaped human ES cell-like colonies emerged in serum-free primate ES cell medium (supplemented with bFGF) in 20% O2 culture conditions. The copy numbers of SeV TS7 vectors in the cytoplasm were drastically reduced by a temperature shift at 38°C for three days. Then, single cells from colonies were seeded on PronectinF?-coated 96-well plates and cultured under na?ve culture conditions (N2B27-based medium supplemented with LIF, forskolin, a MAPK inhibitor, and a GSK inhibitor in 5% O2) for cloning purpose. Dome-shaped mouse ES cell-like colonies from single cells emerged on PronectinF?-coated dishes. These cells were collected and cultured again in primate ES cell medium supplemented with bFGF in 20% O2 and maintained on PronectinF?-coated dishes. Cells were assessed for reprogramming, including the absence of residual SeV and their potential for three germ layer differentiation. Generation of virus-free induced pluripotent stem cell (iPSC) clones from single cells under feeder-free conditions will solve some of the safety concerns related to use of xeno- or allogeneic-material in culture, and contribute to the characterization and the standardization of iPS cells intended for use in a clinical setting.     

The aging signature: a hallmark of induced pluripotent stem cells?

The discovery that somatic cells can be induced into a pluripotent state by the expression of reprogramming factors has enormous potential for therapeutics and human disease modeling. With regard to aging and rejuvenation, the reprogramming process resets an aged, somatic cell to a more youthful state, elongating telomeres, rearranging the mitochondrial network, reducing oxidative stress, restoring pluripotency, and making numerous other alterations. The extent to which induced pluripotent stem cell (iPSC)s mime embryonic stem cells is controversial, however, as iPSCs have been shown to harbor an epigenetic memory characteristic of their tissue of origin which may impact their differentiation potential. Furthermore, there are contentious data regarding the extent to which telomeres are elongated, telomerase activity is reconstituted, and mitochondria are reorganized in iPSCs. Although several groups have reported that reprogramming efficiency declines with age and is inhibited by genes upregulated with age, others have successfully generated iPSCs from senescent and centenarian cells. Mixed findings have also been published regarding whether somatic cells generated from iPSCs are subject to premature senescence. Defects such as these would hinder the clinical application of iPSCs, and as such, more comprehensive testing of iPSCs and their potential aging signature should be conducted.     

“The state of the heart”: Recent advances in engineering human cardiac tissue from pluripotent stem cells

The pressing need for effective cell therapy for the heart has led to the investigation of suitable cell sources for tissue replacement. In recent years, human pluripotent stem cell research expanded tremendously, in particular since the derivation of human-induced pluripotent stem cells. In parallel, bioengineering technologies have led to novel approaches for in vitro cell culture. The combination of these two fields holds potential for in vitro generation of high-fidelity heart tissue, both for basic research and for therapeutic applications. However, this new multidisciplinary science is still at an early stage. Many questions need to be answered and improvements need to be made before clinical applications become a reality. Here we discuss the current status of human stem cell differentiation into cardiomyocytes and the combined use of bioengineering approaches for cardiac tissue formation and maturation in developmental studies, disease modeling, drug testing, and regenerative medicine.