Our laboratory at the Stanford Institute for Stem Cell Biology & Regenerative Medicine and the Department of Developmental Biology aspires to understand how different human cell-types develop from stem cells, and how developing tissues incipiently take shape and form. To this end, we have delineated a comprehensive roadmap that describes how embryonic stem cells can develop into a spectrum of over twenty different human cell types. This roadmap enabled us to generate rather uniform populations of human liver progenitors, bone progenitors and heart progenitors from embryonic stem cells, each of which could regenerate their cognate tissue upon injection into respective mouse models. This platform to produce these engraftable human tissue progenitors provides fundamental building blocks for regenerative medicine and provides an ideal venue to understand human developmental biology. In particular we are interested in questions regarding cellular signaling, developmental competence and tissue organization.
Kyle received his B.A. from Rutgers University, interned with Bing Lim at the Genome Institute of Singapore, and received his Ph.D. from Stanford University (working with Irving Weissman), with fellowships from the Hertz Foundation, the National Science Foundation and the Davidson Institute of Talent Development. He then continued research as the Siebel Investigator at the Stanford Institute for Stem Cell Biology & Regenerative Medicine, and later, as an Assistant Professor of Developmental Biology and The Anthony DiGenova Endowed Faculty Scholar. Kyle is a Packard Fellow, Pew Scholar, Human Frontier Science Program Young Investigator and Baxter Foundation Faculty Scholar, and his research has been recognized by the NIH Director's Early Independence Award, Forbes 30 Under 30, Harold Weintraub Graduate Award, Hertz Foundation Thesis Prize and the A*STAR Investigatorship.
Member, Stanford Diabetes Research Center (2018 - Present)
Siebel Investigator, Stanford Institute for Stem Cell Biology & Regenerative Medicine (2016 - 2018)
Scientific Advisory Board, Americans for Cures Foundation (2015 - Present)
Honors & Awards
Packard Fellowship for Science and Engineering, David and Lucile Packard Foundation (2019)
Pew Scholar, The Pew Charitable Trusts (2019)
Human Frontier Science Program Young Investigator, International Human Frontier Science Program (2019)
Forbes 30 Under 30, Forbes Magazine (2018)
The Anthony DiGenova Endowed Faculty Scholar, Stanford University (2018)
Donald and Delia Baxter Foundation Faculty Scholar, Baxter Foundation (2018)
NIH Director's Early Independence Award DP5, U.S. National Institutes of Health (2017-2022)
Siebel Investigatorship, Stanford School of Medicine (2016)
A*STAR Investigatorship, Singapore Agency for Science, Technology & Research (A*STAR) (2016)
Harold Weintraub Graduate Student Award, Fred Hutchinson Cancer Research Center (2015)
Hertz Foundation Graduate Fellowship Award, The Fannie and John Hertz Foundation (2011)
NSF Graduate Research Fellowship, U.S. National Science Foundation (2011)
Davidson Laureate Fellowship, Davidson Institute for Talent Development (2010)
Harvard Stem Cell Institute Internship Program, Harvard Stem Cell Institute (2008)
Rutgers University School of Arts & Sciences Excellence Award, Rutgers University (2007-2010)
Research & Development Council of New Jersey Scholarship, New Jersey Research & Development Council (2007)
Current Research and Scholarly Interests
Embryonic stem cells can produce any type of human cell in a dish. Thus they afford an opportunity to recreate, and thus study, basic developmental phenomena (lineage diversification, tissue self-organization and multilineage competence) that are difficult to probe in a developing embryo. However, this opportunity has yet to be fully realized because stem-cell differentiation often yields heterogeneous mixtures of cells that are ill-suited for molecular analysis or cell therapy.
We have developed a reductionist system to define the minimal essential inductive and repressive signals necessary for the developmental induction of a given embryonic lineage from differentiating ESCs. These efforts culminated in systematic roadmaps describing the extrinsic signals that guide human ESCs into a variety of endoderm and mesoderm germ layer derivatives (including liver, intestinal, bone and heart progenitors) through a series of bifurcating intermediate steps. The overarching goal is to exploit the resultant highly-pure populations of human tissue progenitors to explore classic questions in developmental biology, using stem-cell differentiation as a technological platform.
- Stem Cell Intensive
STEMREM 200 (Aut)
- Stem Cells and Human Development: From Embryo to Cell Lineage Determination
STEMREM 201A (Aut)
Independent Studies (6)
- Directed Reading in Developmental Biology
DBIO 299 (Aut, Win, Spr, Sum)
- Graduate Research
DBIO 399 (Aut, Win, Spr, Sum)
- Graduate Research
STEMREM 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
DBIO 370 (Aut, Win, Spr, Sum)
- Undergraduate Research
DBIO 199 (Aut, Win, Spr, Sum)
- Undergraduate Research
STEMREM 199 (Spr, Sum)
- Directed Reading in Developmental Biology
Prior Year Courses
- Stem Cell Intensive
STEMREM 200 (Aut)
- Stem Cells and Human Development: From Embryo to Cell Lineage Determination
STEMREM 201A (Aut)
- Stem Cell Intensive
Generating Cellular Diversity and Spatial Form: Wnt Signaling and the Evolution of Multicellular Animals.
2016; 38 (6): 643-655
There were multiple prerequisites to the evolution of multicellular animal life, including the generation of multiple cell fates ("cellular diversity") and their patterned spatial arrangement ("spatial form"). Wnt proteins operate as primordial symmetry-breaking signals. By virtue of their short-range nature and their capacity to activate both lineage-specifying and cell-polarizing intracellular signaling cascades, Wnts can polarize cells at their site of contact, orienting the axis of cell division while simultaneously programming daughter cells to adopt diverging fates in a spatially stereotyped way. By coupling cell fate to position, symmetry-breaking Wnt signals were pivotal in constructing the metazoan body by generating cellular diversity and spatial form.
View details for DOI 10.1016/j.devcel.2016.08.011
View details for PubMedID 27676437
Mapping the Pairwise Choices Leading from Pluripotency to Human Bone, Heart, and Other Mesoderm Cell Types
2016; 166 (2): 451-467
Stem-cell differentiation to desired lineages requires navigating alternating developmental paths that often lead to unwanted cell types. Hence, comprehensive developmental roadmaps are crucial to channel stem-cell differentiation toward desired fates. To this end, here, we map bifurcating lineage choices leading from pluripotency to 12 human mesodermal lineages, including bone, muscle, and heart. We defined the extrinsic signals controlling each binary lineage decision, enabling us to logically block differentiation toward unwanted fates and rapidly steer pluripotent stem cells toward 80%-99% pure human mesodermal lineages at most branchpoints. This strategy enabled the generation of human bone and heart progenitors that could engraft in respective in vivo models. Mapping stepwise chromatin and single-cell gene expression changes in mesoderm development uncovered somite segmentation, a previously unobservable human embryonic event transiently marked by HOPX expression. Collectively, this roadmap enables navigation of mesodermal development to produce transplantable human tissue progenitors and uncover developmental processes. VIDEO ABSTRACT.
View details for DOI 10.1016/j.cell.2016.06.011
View details for PubMedID 27419872
Ex uno plures: molecular designs for embryonic pluripotency.
2015; 95 (1): 245-295
Pluripotent cells in embryos are situated near the apex of the hierarchy of developmental potential. They are capable of generating all cell types of the mammalian body proper. Therefore, they are the exemplar of stem cells. In vivo, pluripotent cells exist transiently and become expended within a few days of their establishment. Yet, when explanted into artificial culture conditions, they can be indefinitely propagated in vitro as pluripotent stem cell lines. A host of transcription factors and regulatory genes are now known to underpin the pluripotent state. Nonetheless, how pluripotent cells are equipped with their vast multilineage differentiation potential remains elusive. Consensus holds that pluripotency transcription factors prevent differentiation by inhibiting the expression of differentiation genes. However, this does not explain the developmental potential of pluripotent cells. We have presented another emergent perspective, namely, that pluripotency factors function as lineage specifiers that enable pluripotent cells to differentiate into specific lineages, therefore endowing pluripotent cells with their multilineage potential. Here we provide a comprehensive overview of the developmental biology, transcription factors, and extrinsic signaling associated with pluripotent cells, and their accompanying subtypes, in vitro heterogeneity and chromatin states. Although much has been learned since the appreciation of mammalian pluripotency in the 1950s and the derivation of embryonic stem cell lines in 1981, we will specifically emphasize what currently remains unclear. However, the view that pluripotency factors capacitate differentiation, recently corroborated by experimental evidence, might perhaps address the long-standing question of how pluripotent cells are endowed with their multilineage differentiation potential.
View details for DOI 10.1152/physrev.00001.2014
View details for PubMedID 25540144
Stem cell signaling. An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control.
2014; 346 (6205)
Stem cells fuel tissue development, renewal, and regeneration, and these activities are controlled by the local stem cell microenvironment, the "niche." Wnt signals emanating from the niche can act as self-renewal factors for stem cells in multiple mammalian tissues. Wnt proteins are lipid-modified, which constrains them to act as short-range cellular signals. The locality of Wnt signaling dictates that stem cells exiting the Wnt signaling domain differentiate, spatially delimiting the niche in certain tissues. In some instances, stem cells may act as or generate their own niche, enabling the self-organization of patterned tissues. In this Review, we discuss the various ways by which Wnt operates in stem cell control and, in doing so, identify an integral program for tissue renewal and regeneration.
View details for DOI 10.1126/science.1248012
View details for PubMedID 25278615
Efficient endoderm induction from human pluripotent stem cells by logically directing signals controlling lineage bifurcations.
Cell stem cell
2014; 14 (2): 237-252
Human pluripotent stem cell (hPSC) differentiation typically yields heterogeneous populations. Knowledge of signals controlling embryonic lineage bifurcations could efficiently yield desired cell types through exclusion of alternate fates. Therefore, we revisited signals driving induction and anterior-posterior patterning of definitive endoderm to generate a coherent roadmap for endoderm differentiation. With striking temporal dynamics, BMP and Wnt initially specified anterior primitive streak (progenitor to endoderm), yet, 24 hr later, suppressed endoderm and induced mesoderm. At lineage bifurcations, cross-repressive signals separated mutually exclusive fates; TGF-β and BMP/MAPK respectively induced pancreas versus liver from endoderm by suppressing the alternate lineage. We systematically blockaded alternate fates throughout multiple consecutive bifurcations, thereby efficiently differentiating multiple hPSC lines exclusively into endoderm and its derivatives. Comprehensive transcriptional and chromatin mapping of highly pure endodermal populations revealed that endodermal enhancers existed in a surprising diversity of "pre-enhancer" states before activation, reflecting the establishment of a permissive chromatin landscape as a prelude to differentiation.
View details for DOI 10.1016/j.stem.2013.12.007
View details for PubMedID 24412311
A Precarious Balance: Pluripotency Factors as Lineage Specifiers
CELL STEM CELL
2011; 8 (4): 363-369
Understanding the basis of the unrestricted multilineage differentiation potential of pluripotent cells will be of developmental and translational consequence. We propose that pluripotency transcription factors are lineage specifiers that direct commitment to specific fetal lineages. Individual factors bestow the ability to differentiate into particular cell types, and concomitant expression of multiple lineage specifiers within pluripotent cells enables differentiation into every fetal lineage. Moreover, we speculate that, rather than being an intrinsically stable "ground state," pluripotency is an inherently precarious condition in which rival lineage specifiers continually compete to specify differentiation along mutually exclusive lineages.
View details for DOI 10.1016/j.stem.2011.03.013
View details for Web of Science ID 000289707100008
View details for PubMedID 21474100
- Bridging naive and primed pluripotency. Nature cell biology 2020
- Spatially controlled stem cell differentiation via morphogen gradients: A comparison of static and dynamic microfluidic platforms JOURNAL OF VACUUM SCIENCE & TECHNOLOGY A 2020; 38 (3)
Spatially controlled stem cell differentiation via morphogen gradients: A comparison of static and dynamic microfluidic platforms.
Journal of vacuum science & technology. A, Vacuum, surfaces, and films : an official journal of the American Vacuum Society
2020; 38 (3): 033205
The ability to harness the processes by which complex tissues arise during embryonic development would improve the ability to engineer complex tissuelike constructs in vitro-a longstanding goal of tissue engineering and regenerative medicine. In embryos, uniform populations of stem cells are exposed to spatial gradients of diffusible extracellular signaling proteins, known as morphogens. Varying levels of these signaling proteins induce stem cells to differentiate into distinct cell types at different positions along the gradient, thus creating spatially patterned tissues. Here, the authors describe two straightforward and easy-to-adopt microfluidic strategies to expose human pluripotent stem cells in vitro to spatial gradients of desired differentiation-inducing extracellular signals. Both approaches afford a high degree of control over the distribution of extracellular signals, while preserving the viability of the cultured stem cells. The first microfluidic platform is commercially available and entails static culture, whereas the second microfluidic platform requires fabrication and dynamic fluid exchange. In each platform, the authors first computationally modeled the spatial distribution of differentiation-inducing extracellular signals. Then, the authors used each platform to expose human pluripotent stem cells to a gradient of these signals (in this case, inducing a cell type known as the primitive streak), resulting in a regionalized culture with differentiated primitive streak cells predominately localized on one side and undifferentiated stem cells at the other side of the device. By combining this approach with a fluorescent reporter for differentiated cells and live-cell fluorescence imaging, the authors characterized the spatial and temporal dynamics of primitive streak differentiation within the induced signaling gradients. Microfluidic approaches to create precisely controlled morphogen gradients will add to the stem cell and developmental biology toolkit, and may eventually pave the way to create increasingly spatially patterned tissuelike constructs in vitro.
View details for DOI 10.1116/1.5142012
View details for PubMedID 32255900
View details for PubMedCentralID PMC7093209
Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells.
Journal of visualized experiments : JoVE
The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure-which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults-is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.
View details for DOI 10.3791/58975
View details for PubMedID 31259908
Antibody Conditioning Enables MHC-Mismatched Hematopoietic Stem Cell Transplants and Organ Graft Tolerance.
Cell stem cell
Hematopoietic cell transplantation can correct hematological and immunological disorders by replacing a diseased blood system with a healthy one, but this currently requires depleting a patient's existing hematopoietic system with toxic and non-specific chemotherapy, radiation, or both. Here we report an antibody-based conditioning protocol with reduced toxicity and enhanced specificity for robust hematopoietic stem cell (HSC) transplantation and engraftment in recipient mice. Host pre-treatment with six monoclonal antibodies targeting CD47, Tcells, NK cells, and HSCs followed by donor HSC transplantation enabled stable hematopoietic system reconstitution in recipients with mismatches at half (haploidentical) or all major histocompatibility complex (MHC) genes. This approach allowed tolerance to heart tissue from HSC donor strains in haploidentical recipients, showing potential applications for solid organ transplantation without immune suppression. Fully mismatched chimeric mice developed antibody responses to nominal antigens, showing preserved functional immunity. These findings suggest approaches for transplanting immunologically mismatched HSCs and solid organs with limited toxicity.
View details for DOI 10.1016/j.stem.2019.05.018
View details for PubMedID 31204177
Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation.
Multipotent self-renewing haematopoietic stem cells (HSCs) regenerate the adult blood system after transplantation1, which is a curative therapy for numerous diseases including immunodeficiencies and leukaemias2. Although substantial effort has been applied to identifying HSC maintenance factors through the characterization of the in vivo bone-marrow HSC microenvironment or niche3-5, stable ex vivo HSC expansion has previously been unattainable6,7. Here we describe the development of a defined, albumin-free culture system that supports the long-term ex vivo expansion of functional mouse HSCs. We used a systematic optimization approach, and found that high levels of thrombopoietin synergize with low levels of stem-cell factor and fibronectin to sustain HSC self-renewal. Serum albumin has long been recognized as a major source of biological contaminants in HSC cultures8; we identify polyvinyl alcohol as a functionally superior replacement for serum albumin that is compatible with good manufacturing practice. These conditions afford between 236- and 899-fold expansions of functional HSCs over 1month, although analysis of clonally derived cultures suggests that there is considerable heterogeneity in the self-renewal capacity of HSCs ex vivo. Using this system, HSC cultures that are derived from only 50cells robustly engraft in recipient mice without the normal requirement for toxic pre-conditioning (for example, radiation), which may be relevant for HSC transplantation in humans. These findings therefore have important implications for both basic HSC research and clinical haematology.
View details for DOI 10.1038/s41586-019-1244-x
View details for PubMedID 31142833
A critical look: Challenges in differentiating human pluripotent stem cells into desired cell types and organoids.
Wiley interdisciplinary reviews. Developmental biology
Too many choices can be problematic. This is certainly the case for human pluripotent stem cells (hPSCs): they harbor the potential to differentiate into hundreds of cell types; yet it is highly challenging to exclusively differentiate hPSCs into a single desired cell type. This review focuses on unresolved and fundamental questions regarding hPSC differentiation and critiquing the identity and purity of the resultant cell populations. These are timely issues in view of the fact that hPSC-derived cell populations have or are being transplanted into patients in over 30 ongoing clinical trials. While many in vitro differentiation protocols purport to "mimic development," the exact number and identity of intermediate steps that a pluripotent cell takes to differentiate into a given cell type in vivo remains largely unknown. Consequently, most differentiation efforts inevitably generate a heterogeneous cellular population, as revealed by single-cell RNA-sequencing and other analyses. The presence of unwanted cell types in differentiated hPSC populations does not portend well for transplantation therapies. This provides an impetus to precisely control differentiation to desired ends-for instance, by logically blocking the formation of unwanted cell types or by overexpressing lineage-specifying transcription factors-or by harnessing technologies to selectively purify desired cell types. Conversely, approaches to differentiate three-dimensional "organoids" from hPSCs intentionally generate heterogeneous cell populations. While this is intended to mimic the rich cellular diversity of developing tissues, whether all such organoids are spatially organized in a manner akin to native organs (and thus, whether they fully qualify as organoids) remains to be fully resolved. This article is categorized under: Adult Stem Cells > Tissue Renewal > Regeneration: Stem Cell Differentiation and Reversion Gene Expression > Transcriptional Hierarchies: Cellular Differentiation Early Embryonic Development: Gastrulation and Neurulation.
View details for DOI 10.1002/wdev.368
View details for PubMedID 31746148
Obliterating Obstacles to an Odyssey.
Cell stem cell
2018; 23 (3): 313–15
Why is reprogramming to generate induced pluripotent stem cells (iPSCs) a protracted and inefficient odyssey? In this issue of Cell Stem Cell, Mor etal. (2018) hypothesize that reprogramming factors paradoxically activate and inhibit pluripotency gene expression and show that eliminating Gatad2a (a NuRD corepressor complex subcomponent) rapidly and efficiently reprograms multiple cell types into iPSCs.
View details for PubMedID 30193127
A Roadmap for Human Liver Differentiation from Pluripotent Stem Cells
2018; 22 (8): 2190–2205
How are closely related lineages, including liver, pancreas, and intestines, diversified from a common endodermal origin? Here, we apply principles learned from developmental biology to rapidly reconstitute liver progenitors from human pluripotent stem cells (hPSCs). Mapping the formation of multiple endodermal lineages revealed how alternate endodermal fates (e.g., pancreas and intestines) are restricted during liver commitment. Human liver fate was encoded by combinations of inductive and repressive extracellular signals at different doses. However, these signaling combinations were temporally re-interpreted: cellular competence to respond to retinoid, WNT, TGF-β, and other signals sharply changed within 24 hr. Consequently, temporally dynamic manipulation of extracellular signals was imperative to suppress the production of unwanted cell fates across six consecutive developmental junctures. This efficiently generated 94.1% ± 7.35% TBX3+HNF4A+ human liver bud progenitors and 81.5% ± 3.2% FAH+ hepatocyte-like cells by days 6 and 18 of hPSC differentiation, respectively; the latter improved short-term survival in the Fah-/-Rag2-/-Il2rg-/- mouse model of liver failure.
View details for PubMedID 29466743
- Isolation and 3D expansion of multipotent Sox9(+) mouse lung progenitors NATURE METHODS 2017; 14 (12): 1205-+
Evaluating the regenerative potential and functionality of human liver cells in mice
2017; 98: 25–34
Liver diseases afflict millions of patients worldwide. Currently, the only long-term treatment for liver failure is the transplantation of a new liver. However, intravenously transplanting a suspension of human hepatocytes might be a less-invasive approach to partially reconstitute lost liver functions in human patients as evinced by promising outcomes in clinical trials. The purpose of this essay is to emphasize outstanding questions that continue to surround hepatocyte transplantation. While adult primary human hepatocytes are the gold standard for transplantation, hepatocytes are heterogeneous. Whether all hepatocytes engraft equally and what specifically defines an "engraftable" hepatocyte capable of long-term liver reconstitution remains unclear. To this end, mouse models of liver injury enable the evaluation of human hepatocytes and their behavior upon transplantation into a complex injured liver environment. While mouse models may not be fully representative of the injured human liver and human hepatocytes tend to engraft mice less efficiently than mouse hepatocytes, valuable lessons have nonetheless been learned from transplanting human hepatocytes into mouse models. With an eye to the future, it will be crucial to eventually detail the optimal biological source (whether in vivo- or in vitro-derived) and presumptive heterogeneity of human hepatocytes and to understand the mechanisms through which they engraft and regenerate liver tissue in vivo.
View details for PubMedID 29078082
Live Imaging Reveals that the First Division of Differentiating Human Embryonic Stem Cells Often Yields Asymmetric Fates.
2017; 21 (2): 301–7
How do stem cells respond to signals to initiate differentiation? Here, we show that, despite uniform exposure to differentiation-inducing extracellular signals, individual human embryonic stem cells (hESCs) respond heterogeneously. To track how hESCs incipiently exit pluripotency, we established a system to differentiate hESCs as single cells and conducted live imaging to track their very first cell division. We followed the fate of their earliest daughters as they remained undifferentiated or differentiated toward the primitive streak (the earliest descendants of pluripotent cells). About 30%-50% of the time, hESCs divided to yield one primitive streak and one undifferentiated daughter. The undifferentiated daughter cell was innately resistant to WNT signaling and could not respond to this primitive-streak-specifying differentiation signal. Hence, the first division of differentiating hESCs sometimes yields daughters with diverging fates, with implications for the efficiency of directed differentiation protocols and the underlying rules of lineage commitment.
View details for PubMedID 29020617
Isolation and 3D expansion of multipotent Sox9+ mouse lung progenitors.
2017; 14 (12): 1205–12
Multiple adult tissues are maintained by stem cells of restricted developmental potential which can only form a subset of lineages within the tissue. For instance, the two adult lung epithelial compartments (airways and alveoli) are separately maintained by distinct lineage-restricted stem cells. A challenge has been to obtain multipotent stem cells and/or progenitors that can generate all epithelial cell types of a given tissue. Here we show that mouse Sox9+ multipotent embryonic lung progenitors can be isolated and expanded long term in 3D culture. Cultured Sox9+ progenitors transcriptionally resemble their in vivo counterparts and generate both airway and alveolar cell types in vitro. Sox9+ progenitors that were transplanted into injured adult mouse lungs differentiated into all major airway and alveolar lineages in vivo in a region-appropriate fashion. We propose that a single expandable embryonic lung progenitor population with broader developmental competence may eventually be used as an alternative for region-restricted adult tissue stem cells in regenerative medicine.
View details for PubMedID 29106405
Thirst-associated preoptic neurons encode an aversive motivational drive.
Science (New York, N.Y.)
2017; 357 (6356): 1149–55
Water deprivation produces a drive to seek and consume water. How neural activity creates this motivation remains poorly understood. We used activity-dependent genetic labeling to characterize neurons activated by water deprivation in the hypothalamic median preoptic nucleus (MnPO). Single-cell transcriptional profiling revealed that dehydration-activated MnPO neurons consist of a single excitatory cell type. After optogenetic activation of these neurons, mice drank water and performed an operant lever-pressing task for water reward with rates that scaled with stimulation frequency. This stimulation was aversive, and instrumentally pausing stimulation could reinforce lever-pressing. Activity of these neurons gradually decreased over the course of an operant session. Thus, the activity of dehydration-activated MnPO neurons establishes a scalable, persistent, and aversive internal state that dynamically controls thirst-motivated behavior.
View details for PubMedID 28912243
An atlas of transcriptional, chromatin accessibility, and surface marker changes in human mesoderm development
Mesoderm is the developmental precursor to myriad human tissues including bone, heart, and skeletal muscle. Unravelling the molecular events through which these lineages become diversified from one another is integral to developmental biology and understanding changes in cellular fate. To this end, we developed an in vitro system to differentiate human pluripotent stem cells through primitive streak intermediates into paraxial mesoderm and its derivatives (somites, sclerotome, dermomyotome) and separately, into lateral mesoderm and its derivatives (cardiac mesoderm). Whole-population and single-cell analyses of these purified populations of human mesoderm lineages through RNA-seq, ATAC-seq, and high-throughput surface marker screens illustrated how transcriptional changes co-occur with changes in open chromatin and surface marker landscapes throughout human mesoderm development. This molecular atlas will facilitate study of human mesoderm development (which cannot be interrogated in vivo due to restrictions on human embryo studies) and provides a broad resource for the study of gene regulation in development at the single-cell level, knowledge that might one day be exploited for regenerative medicine.
View details for DOI 10.1038/sdata.2016.109
View details for PubMedID 27996962
Reprogramming mouse fibroblasts into engraftable myeloerythroid and lymphoid progenitors
Recent efforts have attempted to convert non-blood cells into hematopoietic stem cells (HSCs) with the goal of generating blood lineages de novo. Here we show that hematopoietic transcription factors Scl, Lmo2, Runx1 and Bmi1 can convert a developmentally distant lineage (fibroblasts) into 'induced hematopoietic progenitors' (iHPs). Functionally, iHPs generate acetylcholinesterase(+) megakaryocytes and phagocytic myeloid cells in vitro and can also engraft immunodeficient mice, generating myeloerythoid and B-lymphoid cells for up to 4 months in vivo. Molecularly, iHPs transcriptionally resemble native Kit(+) hematopoietic progenitors. Mechanistically, reprogramming factor Lmo2 implements a hematopoietic programme in fibroblasts by rapidly binding to and upregulating the Hhex and Gfi1 genes within days. Moreover the reprogramming transcription factors also require extracellular BMP and MEK signalling to cooperatively effectuate reprogramming. Thus, the transcription factors that orchestrate embryonic hematopoiesis can artificially reconstitute this programme in developmentally distant fibroblasts, converting them into engraftable blood progenitors.
View details for DOI 10.1038/ncomms13396
View details for PubMedID 27869129
Inhibition of Apoptosis Overcomes Stage-Related Compatibility Barriers to Chimera Formation in Mouse Embryos.
Cell stem cell
2016; 19 (5): 587-592
Cell types more advanced in development than embryonic stem cells, such as EpiSCs, fail to contribute to chimeras when injected into pre-implantation-stage blastocysts, apparently because the injected cells undergo apoptosis. Here we show that transient promotion of cell survival through expression of the anti-apoptotic gene BCL2 enables EpiSCs and Sox17(+) endoderm progenitors to integrate into blastocysts and contribute to chimeric embryos. Upon injection into blastocyst, BCL2-expressing EpiSCs contributed to all bodily tissues in chimeric animals while Sox17(+) endoderm progenitors specifically contributed in a region-specific fashion to endodermal tissues. In addition, BCL2 expression enabled rat EpiSCs to contribute to mouse embryonic chimeras, thereby forming interspecies chimeras that could survive to adulthood. Our system therefore provides a method to overcome cellular compatibility issues that typically restrict chimera formation. Application of this type of approach could broaden the use of embryonic chimeras, including region-specific chimeras, for basic developmental biology research and regenerative medicine.
View details for DOI 10.1016/j.stem.2016.10.013
View details for PubMedID 27814480
- Stem cells: Equilibrium established. Nature 2015; 521 (7552): 299-300
Differentiation of trophoblast cells from human embryonic stem cells: to be or not to be?
2014; 147 (5): D1-D12
It is imperative to unveil the full range of differentiated cell types into which human pluripotent stem cells (hPSCs) can develop. The need is twofold: it will delimit the therapeutic utility of these stem cells and is necessary to place their position accurately in the developmental hierarchy of lineage potential. Accumulated evidence suggested that hPSC could develop in vitro into an extraembryonic lineage (trophoblast (TB)) that is typically inaccessible to pluripotent embryonic cells during embryogenesis. However, whether these differentiated cells are truly authentic TB has been challenged. In this debate, we present a case for and a case against TB differentiation from hPSCs. By analogy to other differentiation systems, our debate is broadly applicable, as it articulates higher and more challenging standards for judging whether a given cell type has been genuinely produced from hPSC differentiation.
View details for DOI 10.1530/REP-14-0080
View details for Web of Science ID 000336898100001
- Rapid and Efficient Conversion of Integration-Free Human Induced Pluripotent Stem Cells to GMP-Grade Culture Conditions. PloS one 2014; 9 (4)
Rapid and efficient conversion of integration-free human induced pluripotent stem cells to GMP-grade culture conditions.
2014; 9 (4)
Data suggest that clinical applications of human induced pluripotent stem cells (hiPSCs) will be realized. Nonetheless, clinical applications will require hiPSCs that are free of exogenous DNA and that can be manufactured through Good Manufacturing Practice (GMP). Optimally, derivation of hiPSCs should be rapid and efficient in order to minimize manipulations, reduce potential for accumulation of mutations and minimize financial costs. Previous studies reported the use of modified synthetic mRNAs to reprogram fibroblasts to a pluripotent state. Here, we provide an optimized, fully chemically defined and feeder-free protocol for the derivation of hiPSCs using synthetic mRNAs. The protocol results in derivation of fully reprogrammed hiPSC lines from adult dermal fibroblasts in less than two weeks. The hiPSC lines were successfully tested for their identity, purity, stability and safety at a GMP facility and cryopreserved. To our knowledge, as a proof of principle, these are the first integration-free iPSCs lines that were reproducibly generated through synthetic mRNA reprogramming that could be putatively used for clinical purposes.
View details for DOI 10.1371/journal.pone.0094231
View details for PubMedID 24718618
Clonal precursor of bone, cartilage, and hematopoietic niche stromal cells
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2013; 110 (31): 12643-12648
Organs are composites of tissue types with diverse developmental origins, and they rely on distinct stem and progenitor cells to meet physiological demands for cellular production and homeostasis. How diverse stem cell activity is coordinated within organs is not well understood. Here we describe a lineage-restricted, self-renewing common skeletal progenitor (bone, cartilage, stromal progenitor; BCSP) isolated from limb bones and bone marrow tissue of fetal, neonatal, and adult mice. The BCSP clonally produces chondrocytes (cartilage-forming) and osteogenic (bone-forming) cells and at least three subsets of stromal cells that exhibit differential expression of cell surface markers, including CD105 (or endoglin), Thy1 [or CD90 (cluster of differentiation 90)], and 6C3 [ENPEP glutamyl aminopeptidase (aminopeptidase A)]. These three stromal subsets exhibit differential capacities to support hematopoietic (blood-forming) stem and progenitor cells. Although the 6C3-expressing subset demonstrates functional stem cell niche activity by maintaining primitive hematopoietic stem cell (HSC) renewal in vitro, the other stromal populations promote HSC differentiation to more committed lines of hematopoiesis, such as the B-cell lineage. Gene expression analysis and microscopic studies further reveal a microenvironment in which CD105-, Thy1-, and 6C3-expressing marrow stroma collaborate to provide cytokine signaling to HSCs and more committed hematopoietic progenitors. As a result, within the context of bone as a blood-forming organ, the BCSP plays a critical role in supporting hematopoiesis through its generation of diverse osteogenic and hematopoietic-promoting stroma, including HSC supportive 6C3(+) niche cells.
View details for DOI 10.1073/pnas.1310212110
View details for PubMedID 23858471
- Rejuvenating tithonus. EMBO reports 2013; 14 (7): 583-584
- EPIGENETICS Actors in the cell reprogramming drama NATURE 2012; 488 (7413): 599-600
- Investigating the bona fide differentiation capacity of human pluripotent stem cells CELL RESEARCH 2012; 22 (1): 6-8
CELL STEM CELL
2010; 7 (2): 137-139
Two Matters Arising articles in this issue challenge the conclusions of a previous Cell Stem Cell paper that found extensive transcriptional differences between hESCs and hiPSCs. The original authors provide a response and set in motion a discussion in the field about appropriate methods for microarray data analysis.
View details for DOI 10.1016/j.stem.2010.07.005
View details for Web of Science ID 000281107400002
View details for PubMedID 20682438
A Small-Molecule Inhibitor of Tgf-beta Signaling Replaces Sox2 in Reprogramming by Inducing Nanog
CELL STEM CELL
2009; 5 (5): 491-503
The combined activity of three transcription factors can reprogram adult cells into induced pluripotent stem cells (iPSCs). However, the transgenic methods used for delivering reprogramming factors have raised concerns regarding the future utility of the resulting stem cells. These uncertainties could be overcome if each transgenic factor were replaced with a small molecule that either directly activated its expression from the somatic genome or in some way compensated for its activity. To this end, we have used high-content chemical screening to identify small molecules that can replace Sox2 in reprogramming. We show that one of these molecules functions in reprogramming by inhibiting Tgf-beta signaling in a stable and trapped intermediate cell type that forms during the process. We find that this inhibition promotes the completion of reprogramming through induction of the transcription factor Nanog.
View details for DOI 10.1016/j.stem.2009.09.012
View details for Web of Science ID 000272019500011
View details for PubMedID 19818703