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The International Society for Stem Cell Research (ISSCR) presents its 2016 Guidelines for Stem Cell Research and Clinical Translation (ISSCR, 2016). The 2016 guidelines reflect the revision and extension of two past sets of guidelines (ISSCR, 2006; ISSCR, 2008) to address new and emerging areas of stem cell discovery and application and evolving ethical, social, and policy challenges. These guidelines provide an integrated set of principles and best practices to drive progress in basic, translational, and clinical research. The guidelines demand rigor, oversight, and transparency in all aspects of practice, providing confidence to practitioners and public alike that stem cell science can proceed efficiently and remain responsive to public and patient interests. Here, we highlight key elements and recommendations in the guidelines and summarize the recommendations and deliberations behind them., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Stem cells are but one class of the myriad types of cells within an organism. With potential to self-renew and capacity to differentiate, stem cells play essential roles at multiple stages of development. In the early embryo, pluripotent stem cells represent progenitors for all tissues while later in development, tissue-restricted stem cells give rise to cells with highly specialized functions. As best understood in the blood, skin and gut, stem cells are the seeds that sustain tissue homeostasis and regeneration, while in other tissues like the muscle, liver, kidney and lung, various stem or progenitor cells play facultative roles in tissue repair and response to injury. Here, I will provide a brief perspective on the evolving notion of cellular identity and how reprogramming and transcription factor-mediated conversions of one cell type into another have fundamentally altered our assumptions about the stability of cell identity, with profound long-term implications for biomedical research and regenerative medicine., (© 2015 The Author(s).)

Somatic cell reprogramming, directed differentiation of pluripotent stem cells, and direct conversions between differentiated cell lineages represent powerful approaches to engineer cells for research and regenerative medicine. We have developed CellNet, a network biology platform that more accurately assesses the fidelity of cellular engineering than existing methodologies and generates hypotheses for improving cell derivations. Analyzing expression data from 56 published reports, we found that cells derived via directed differentiation more closely resemble their in vivo counterparts than products of direct conversion, as reflected by the establishment of target cell-type gene regulatory networks (GRNs). Furthermore, we discovered that directly converted cells fail to adequately silence expression programs of the starting population and that the establishment of unintended GRNs is common to virtually every cellular engineering paradigm. CellNet provides a platform for quantifying how closely engineered cell populations resemble their target cell type and a rational strategy to guide enhanced cellular engineering., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Wilms Tumor, the most common pediatric kidney cancer, evolves from the failure of terminal differentiation of the embryonic kidney. Here we show that overexpression of the heterochronic regulator Lin28 during kidney development in mice markedly expands nephrogenic progenitors by blocking their final wave of differentiation, ultimately resulting in a pathology highly reminiscent of Wilms tumor. Using lineage-specific promoters to target Lin28 to specific cell types, we observed Wilms tumor only when Lin28 is aberrantly expressed in multiple derivatives of the intermediate mesoderm, implicating the cell of origin as a multipotential renal progenitor. We show that withdrawal of Lin28 expression reverts tumorigenesis and markedly expands the numbers of glomerulus-like structures and that tumor formation is suppressed by enforced expression of Let-7 microRNA. Finally, we demonstrate overexpression of the LIN28B paralog in a significant percentage of human Wilms tumor. Our data thus implicate the Lin28/Let-7 pathway in kidney development and tumorigenesis.

In recent years, the highly conserved Lin28 RNA-binding proteins have emerged as factors that define stemness in several tissue lineages. Lin28 proteins repress let-7 microRNAs and influence mRNA translation, thereby regulating the self-renewal of mammalian embryonic stem cells. Subsequent discoveries revealed that Lin28a and Lin28b are also important in organismal growth and metabolism, tissue development, somatic reprogramming, and cancer. In this review, we discuss the Lin28 pathway and its regulation, outline its roles in stem cells, tissue development, and pathogenesis, and examine the ramifications for re-engineering mammalian physiology., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Stem cells are the seeds of tissue repair and regeneration and a promising source for novel therapies. However, apart from hematopoietic stem cell (HSC) transplantation, essentially all other stem cell treatments remain experimental. High hopes have inspired numerous clinical trials, but it has been difficult to obtain unequivocal evidence for robust clinical benefit. In recent years, unproven therapies have been widely practiced outside the standard clinical trial network, threatening the cause of legitimate clinical investigation. Numerous challenges and technical barriers must be overcome before novel stem cell therapies can achieve meaningful clinical impact., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Over the last decade, a remarkable number of papers have been published in which the biology of stem cells is introduced with words and phrases such as "promise," "rapid progress," and "future therapies." To separate myth and hype from reality, the articles in this Stem Cells Review series comprise a rich resource on the state of this fast-paced field and provide a balanced perspective on some of the major advances. They recount what the field has achieved over the past decade and where the field is headed. They also highlight the challenges to be faced in translating what is indeed highly promising science into proven therapies that will regenerate and repair diseased tissues.

Hemogenic endothelium has been identified in embryonic dorsal aorta and in tissues generated from mouse embryonic stem cells, but to date there is no evidence for such bipotential cells in postnatal tissues or blood. Here we identify a cell population from human umbilical cord blood that gives rise to both endothelial cells and hematopoietic progenitors in vitro. Cord blood CD34+/CD133+ cells plated at high density in an endothelial basal medium formed an endothelial monolayer and a nonadherent cell population after 14-21 days. AML-1, a factor required for definitive hematopoiesis, was detected at low levels in adherent cells and at high levels in nonadherent cells. Nonadherent cells coexpressed the endothelial marker vascular endothelial (VE)-cadherin and the hematopoietic marker CD45, whereas adherent cells were composed primarily of VE-cadherin+/CD45- cells and a smaller fraction of VE-cadherin+/CD45+ cells. Both nonadherent and adherent cells produced hematopoietic colonies in methylcellulose, with the adherent cells yielding more colony-forming units (CFU)-GEMM compared with the nonadherent cells. To determine whether the adherent endothelial cells were producing hematopoietic progenitors, single cells from the adherent population were expanded in 96-well dishes for 14 days. The clonal populations expressed VE-cadherin, and a subset expressed AML-1, epsilon-globin, and gamma-globin. Three of 17 clonal cell populations gave rise to early CFU-GEMM hematopoietic progenitors and burst-forming unit-erythroid progenitors. These results provide evidence for hemogenic endothelial cells in human umbilical cord blood.

Our considerable wealth of data concerning hematologic processes has come despite difficulties working with stem and progenitor cells in vitro and their propensity to differentiate. Key methodologies that have sought to overcome such limitations include transgenic/knock-out animals and in vitro studies using murine embryonic stem cells, because both permit investigation of the formation of hematopoietic tissue from nonhematopoietic precursors. Although there have been many successful studies in model animals for understanding hematopoietic-cell development, differences between lower vertebrates and humans have left gaps in our understanding. Clearly, human-specific strategies to study the onset of hematopoiesis, particularly the earliest events leading to the specification of both normal and abnormal hematopoietic tissue, could bring an investigational renaissance. The recent availability of human embryonic stem (hES) cells suggests that such a system is now at hand. This review highlights the potential of hES cells to model human hematologic processes in vitro with an emphasis on disease targets.

Embryonic stem cells provide an in vitro model for developmental biologists to study cell fate decisions during ontogenesis, while somatic stem cells allow physiologists to understand tissue homeostasis in the adult. The behavior of stem cells is dependent on an intimate relationship with a supportive niche. This brief review highlights some of the most important recent trends in stem cell biology, focusing in particular on the supportive microenvironments for both embryonic and adult stem cells. Known intrinsic and extrinsic molecular players from the best-characterized stem cell types are summarized, illuminating a number of shared environmental cues among tissues originating from all three embryonic germ layers.

Despite two decades of studies documenting the in vitro blood-forming potential of murine embryonic stem cells (ESCs), achieving stable long-term blood engraftment of ESC-derived hematopoietic stem cells in irradiated mice has proven difficult. We have exploited the Cdx-Hox pathway, a genetic program important for blood development, to enhance the differentiation of ESCs along the hematopoietic lineage. Using an embryonic stem cell line engineered with tetracycline-inducible Cdx4, we demonstrate that ectopic Cdx4 expression promotes hematopoietic mesoderm specification, increases hematopoietic progenitor formation, and, together with HoxB4, enhances multilineage hematopoietic engraftment of lethally irradiated adult mice. Clonal analysis of retroviral integration sites confirms a common stem cell origin of lymphoid and myeloid populations in engrafted primary and secondary mice. These data document the cardinal stem cell features of self-renewal and multilineage differentiation of ESC-derived hematopoietic stem cells.

Nearly 20 years after murine embryonic stem cells (mESC) were isolated, the first report of the derivation of human embryonic stem cells (hESC) in 1998 spawned the field of hESC research [Evans MJ, Kaufman MH, Establishment in culture of pluripotential cells from mouse embryos. Nature 1981; 292 (5819): 154-6; Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts. Science 1998; 282 (5391): 1145-7.]. Although this field is only in its infancy, hESC represent a theoretically inexhaustible source of precursor cells that could be differentiated into any cell type to treat degenerative, malignant, or genetic diseases, or injury due to inflammation, infection, and trauma. This pluripotent, endlessly dividing cell has been hailed as a possible means for treating diabetes, Parkinson's disease, Alzheimer's, spinal cord injury, heart failure, and bone marrow failure. But the regenerative medicine applications of embryonic stem cells are only one facet of hESC therapeutic potential. Human ESC are an invaluable research tool to study development, both normal and abnormal, and can serve as a platform to develop and test new therapies. In addition to discussing the therapeutic potential of hESC, this chapter will cover limitations to using hESC for replacement cell therapy, strategies to overcome these limitations, and alternative methods of deriving hESC.

In this report we have investigated the role of the homeobox gene Hex in the development and differentiation of the blast colony-forming cell (BL-CFC), a progenitor with hemangioblast characteristics generated in embryonic stem (ES) cell-derived embryoid bodies (EBs). Molecular analysis showed that Hex is expressed in mesoderm, in populations that contain BL-CFCs, and in blast cell colonies, the progeny of the BL-CFCs. Hex(-/-) EBs displayed a defect in macrophage development but generated higher numbers of BL-CFCs than did wild-type EBs. In addition to differences in these progenitor populations, we also found that endothelial cells from the Hex(-/-) EBs showed enhanced proliferative potential compared with those from wild-type EBs. Forced expression of Hex at the onset of ES cell differentiation resulted in reduced EB cellularity, fetal liver kinase-1 (Flk-1) expression, and BL-CFC development. Taken together, these findings demonstrate that Hex functions at multiple stages of development within the differentiating EBs and uncover a novel role for this transcription factor as a negative regulator of the hemangioblast and the endothelial lineage.

Motivation: Signaling events that direct mouse embryonic stem (ES) cell self-renewal and differentiation are complex and accordingly difficult to understand in an integrated manner. We address this problem by adapting a Bayesian network learning algorithm to model proteomic signaling data for ES cell fate responses to external cues. Using this model we were able to characterize the signaling pathway influences as quantitative, logic-circuit type interactions. Our experimental dataset includes measurements for 28 signaling protein phosphorylation states across 16 different factorial combinations of cytokine and matrix stimuli as reported previously., Results: The Bayesian network modeling approach allows us to uncover previously reported signaling activities related to mouse ES cell self-renewal, such as the roles of LIF and STAT3 in maintaining undifferentiated ES cell populations. Furthermore, the network predicts novel influences such as between ERK phosphorylation and differentiation, or RAF phosphorylation and differentiated cell proliferation. Visualization of the influences detected by the Bayesian network provides intuition about the underlying physiology of the signaling pathways. We demonstrate that the Bayesian networks can capture the linear, nonlinear and multistate logic interactions that connect extracellular cues, intracellular signals and consequent cell functional responses.

Many insights into mammalian germ cell development have been gained through genetic engineering and in vivo studies, but the lack of an in vitro system for deriving germ cells has hindered potential advances in germ cell biology. Recent studies have demonstrated embryonic stem cell differentiation into germ cells and more mature gametes, although significant unanswered questions remain about the functionality of these cells. The derivation of germ cells from embryonic stem cells in vitro provides an invaluable assay both for the genetic dissection of germ cell development and for epigenetic reprogramming, and may one day facilitate nuclear transfer technology and infertility treatments.

In thinking about the practical application of stem cell biology to clinical situations--particularly for the central nervous system (CNS)-it is instructive to remember that the neural stem cell (NSC) field--as a prototype for somatic stem cells in general-emerged as the unanticipated byproduct of investigations by developmental neurobiologists into fundamental aspects of neural determination, commitment, and plasticity. Stem cell behavior is ultimately an expression of developmental principles, an alluring vestige from the more plastic and generative stages of organogenesis. In attempting to apply stem cell biology therapeutically, it is instructive always to bear in mind what role the stem cell plays in development and to what cues it was "designed" to respond in trying to understand the "logic" behind its behavior (both what investigators want to see and what investigators do not want to see). Furthermore, in transplantation paradigms, the interaction between engrafted NSCs and recipient host is a dynamic, complex, ongoing reciprocal interaction where both entities are constantly in flux. In this review, we propose a "roadmap" to the clinic, with a particular emphasis on flagging the "potholes" and "speed bumps" through which we must navigate. Despite the admonitions to be circumspect, we also suggest disease processes that may be within the grasp of proven stem cell properties and might be approachable in the relatively near future., (Copyright 2004 Wiley-Liss, Inc.)

A number of extracellular stimuli, including soluble cytokines and insoluble matrix factors, are known to influence murine embryonic stem cell self-renewal and differentiation behavioral responses via intracellular signaling pathways, but their net effects in combination are difficult to understand. To gain insight concerning key intracellular signals governing these behavioral responses, we employ a multivariate systems analysis of proteomic data generated from combinatorial stimulation of mouse embryonic stem cells by fibronectin, laminin, leukemia-inhibitory factor, and fibroblast growth factor 4. Phosphorylation states of 31 intracellular signaling network components were obtained across 16 different stimulus conditions at three time points by quantitative Western blotting, and partial-least-squares modeling was used to determine which components were most strongly correlated with cell proliferation and differentiation rate constants obtained from flow cytometry measurements of Oct-4 expression levels. This data-driven, multivariate (16 conditions x 31 components x 3 time points = approximately 1,500 values) proteomic approach identified a set of signaling network components most critically associated (positively or negatively) with differentiation (Stat3, Raf1, MEK, and ERK), proliferation of undifferentiated cells (MEK and ERK), and proliferation of differentiated cells (PKB alpha, Stat3, Src, and PKC epsilon). These predictions were found to be consistent with previous in vivo literature, along with direct in vitro test here by a peptide inhibitor of PKC epsilon. Our results demonstrate how a computational systems biology approach can elucidate key sets of intracellular signaling protein activities that combine to govern cell phenotypic responses to extracellular cues.

Egg and sperm cells (gametes) of the mouse are derived from a founder population of primordial germ cells that are set aside early in embryogenesis. Primordial germ cells arise from the proximal epiblast, a region of the early mouse embryo that also contributes to the first blood lineages of the embryonic yolk sac. Embryonic stem cells differentiate in vitro into cystic structures called embryoid bodies consisting of tissue lineages typical of the early mouse embryo. Because embryoid bodies sustain blood development, we reasoned that they might also support primordial germ cell formation. Here we isolate primordial germ cells from embryoid bodies, and derive continuously growing lines of embryonic germ cells. Embryonic germ cells show erasure of the methylation markers (imprints) of the Igf2r and H19 genes, a property characteristic of the germ lineage. We show that embryoid bodies support maturation of the primordial germ cells into haploid male gametes, which when injected into oocytes restore the somatic diploid chromosome complement and develop into blastocysts. Our ability to derive germ cells from embryonic stem cells provides an accessible in vitro model system for studies of germline epigenetic modification and mammalian gametogenesis.

Though a topic of medical interest for centuries, our understanding of vertebrate hematopoietic or "blood-forming" tissue development has improved greatly only in recent years and given a series of scientific and technical milestones. Key among these observations was the description of procedures that allowed the transplantation of blood-forming activity. Beyond this, other advances include the creation of a variety of knock-out animals (mice and more recently zebrafish), microdissection of embryonic and fetal blood-forming tissues, hematopoietic stem (HSC) and progenitor cell (HPC) colony-forming assays, the discovery of cytokines with defined hematopoietic activities, gene transfer technologies, and the description of lineage-specific surface antigens for the identification and purification of pluripotent and differentiated blood cells. The availability of both murine and human embryonic stem cells (ESC) and the delineation of in vitro systems to direct their differentiation have now been added to this analytical arsenal. Such tools have allowed researchers to interrogate the complex developmental processes behind both primitive (yolk sac or extraembryonic) and definitive (intraembryonic) hematopoietic tissue formation. Using ES cells, we hope to not only gain additional basic insights into hematopoietic development but also to develop platforms for therapeutic use in patients suffering from hematological disease. In this review, we will focus on points of convergence and divergence between murine and human hematopoiesis in vivo and in vitro, and use these observations to evaluate the literature regarding attempts to create hematopoietic tissue from embryonic stem cells, the pitfalls encountered therein, and what challenges remain.

Differentiation of embryonic stem (ES) cells in vitro yields abundant hematopoietic progenitors, but achieving stable hematopoietic engraftment of irradiated mice has proven difficult, begging the question of whether ES cells give rise to hematopoietic stem cells in vitro, and limiting the application of ES cells as experimental and therapeutic models. We have employed a number of hematopoietic regulatory genes to probe the nature and developmental potential of ES-derived blood precursors. The chronic myeloid leukemia-associated BCR/ABL oncoprotein transforms a novel class of ES-derived embryonic hematopoietic stem cell that represents a common progenitor of primitive erythropoiesis and definitive lymphoid-myeloid blood development. Expression of the homeobox gene HoxB4 generated normal, non-leukemic hematopoietic progenitors that enabled long-term, multilineage hematopoietic engraftment in primary and secondary mouse recipients. We have used these repopulating hematopoietic stem cells to model therapeutic transplantation from ES cells. We treated an immunodeficient Rag2(-/-) mouse by therapeutic cloning, that is, isogenic ES cell generation by somatic cell nuclear transfer, gene correction, and cell replacement therapy. Comparable approaches with human ES cells are being developed to lay the foundation for cellular therapies in patients with a variety of bone marrow diseases.

With common scientific themes and experimental strategies, stem cell biology is evolving into a recognizable discipline. Its clinical arm, regenerative medicine, is also gaining momentum-invigorated by the potential of stem cells to provide treatments for a host of medical conditions that are poorly served by drug therapy. But are the expectations for stem cell therapies realistic or overstated? In the past year, neurons, insulin-producing cells, and hematopoietic stem cells have been generated from embryonic stem cells or cultivated from somatic tissues of the adult. These cells have yielded modest and preliminary hints of functional reconstitution in animal models. Although encouraging, significant hurdles remain before the promise of stem cells will be realized in the clinic.

A major limitation of the widespread use of stem cells in a variety of biotechnological applications is the relatively low level of knowledge about how to maintain these cells in vitro without losing the long-term multilineage growth properties required for their clinical utility. An experimental and theoretical framework for predicting and controlling the outcome of stem cell stimulation by exogenous cytokines would thus be useful. An emerging theme from recent hematopoietic stem cell (HSC)-expansion studies is that a net gain in HSC numbers requires the maintenance of critical signaling ligand(s) above a threshold level. These ligand-receptor complex thresholds can be maintained, for example, by high concentrations of soluble cytokines or by cytokine presentation on cell surfaces. According to such a model, when the relevant ligand-receptor interaction falls below this threshold level, the probability of a differentiation response is increased; otherwise, self-renewal is favored. Taking advantage of the ability of the cytokine leukemia inhibitory factor (LIF) to maintain embryonic stem (ES) cell pluripotentiality at high concentrations, we are testing this model by investigating critical parameters in the control of ES cell responses. We have developed quantitative assays of ES cell differentiation by measuring cell-surface alkaline phosphatase activity, cell-surface stage specific embryonic antigen (SSEA)-1 expression, and the ability of ES cells to form embryoid bodies. Examination of ES cell responses over a range of LIF concentrations shows that LIF supplementation has little effect on ES cell-growth rate but significantly alters the probability of a cell undergoing a self-renewal vs. a differentiation division. In vitro culture parameters such as inoculum cell density, medium exchange, as well as cell-intrinsic processes such as autocrine secretion are shown to affect this decision. In addition to yielding new information on stem cell regulation by exogenous factors, these studies provide important clues about culture of these cells and should stimulate further investigations into the mechanistic basis of stem cell differentiation control., (Copyright 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 69: 607-617, 2000.)