1987 — 1997 |
Lemischka, Ihor R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
In Vivo Behavior of Hematopoietic Stem Cell Clones
The proposed experiments are aimed at elucidating the in vivo properties of hematopoietic stem cells with respect to developmental potential and temporal organization. The use of retroviral-mediated gene transfer provides a means of making in a unique and benign fashion each stem cell in a bone marrow explant. Genetically transduced bone marrow is used to engraft lethally irradiated recipient mice. Stem cells in a bone marrow inoculum are defined by a quantitative Southern blot analysis of myeloid and lymphoid cell populations isolated from engrafted mice. The existence and proliferative capacity of each stem cell is therefore retroactivity defined by the presence and magnitude of a given integrant in a mature cell population. The developmental potential of such cells is defined by the distribution of an integrant in cell populations of different lineages. Analysis of numerous mice should permit the derivation of a developmental fate-map which may, for the first time in a mammalian system, describe decisions and regulatory phenomena governing a complex program of cellular differentiation. Further studies begin to address the relative importance of intrinsic properties and host environment in the in vivo behavior of stem cell clones. The representation of individual members of a stem cell population as mature cell progeny over time will be assessed by periodic DNA analysis of the blood. This will result in a direct test of clonal succession models which predict that only a subset of the entire stem cell pool is actively engaged in hematopoiesis at any point in time. The experiments will further define kinetic parameters of hematopoiesis in response to artificial stress, thus providing an estimate of the flexibility inherent in this system. Further experiments will attempt to develop systems to allow engraftment with physically isolated stem cell clones. Focusing on in vivo as well as in vitro isolation of such clones, it should be possible to precisely evaluate the properties of the CFU-S as well as to begin interrelating the pluripotent stem cell, the CFU-S and thee clonogenic progenitor cell. The results obtained will provide a framework for future experimentation aimed at a mechanistic understanding of hematopoietic differentiation. The similarity of the murine and human blood systems implies that the proposed experiments will lead to a better understanding of hematopoiesis in man, and thus allow for the design of more effective therapeutic strategies in the treatment of genetic as well as acquired syndromes.
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1990 — 1993 |
Lemischka, Ihor R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Clonal &Molecular Studies of Normal Hematopoiesis
These studies will extend technologies of genetic-marking to an analysis of hematopoietic stem cell clonal behavior in normal non-ablated animals. Such a fate-map of the normal unperturbed hematopoietic system in comparison to ongoing studies utilizing irradiated hosts for stem cell assays will provide a first assessment of cell intrinsic vs. environmental factors in the formation of all blood cell lineages in normal as well as pathological conditions such as leukemia. In addition, these studies should shed light on parameters involved in bone marrow transplantation therapies. In the first strategy, purified retrovirally-marked stem cells will be engrafted into normal and mutant fetal mice by transplacental injection. Such cells should subsequently colonize the bone environment in a normal fashion and at the correct developmental time and contribute to hematopoiesis throughout adult life. A second strategy will combine germ- line transgenesis with somatic retroviral-mediated gene transfer to generate lines of mice that will introduce random stable proviral markers into hematopoietic stem cells as they develop in the normal environment. The key principal is the germ-line introduction of chimeric retroviral transgenes whose expression is targeted to cells comprising the niches within which hematopoietic stem cells proliferate. This will result in local production of marker virus which will infect the stem cells, inactivate itself local production of marker virus which will infect he stem cells, inactivate itself and therefore uniquely and permanently mark the cells and their resultant clonal progeny. The expression of marker virus is designed to occur only in the temporal frame during which stem cells first appear and clonally expand during ontogeny. Sequential and quantitative Southern blot analysis of proviral marker distribution in peripheral blood cell types obtained from animals derived from both approaches will define the developmental and proliferative behavior of the stem cell in its normal environment. The second proposed set of experiments will provide inroads to a precise molecular dissection of regulatory mechanisms which function in proliferation/differentiation decisions of primitive stem cells. Strategies are proposed to analyze gene expression directly in purified stem cells and to isolate stem cell specific members of regulatory gene families. These will provide direct insight into hematopoietic regulation as it functions in normal blood formation and malfunction to yield cancerous or other hematologic disorders.
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1994 |
Lemischka, Ihor R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Clonal and Molecular Studies of Normal Hematopoiesis
These studies will extend technologies of genetic-marking to an analysis of hematopoietic stem cell clonal behavior in normal non-ablated animals. Such a fate-map of the normal unperturbed hematopoietic system in comparison to ongoing studies utilizing irradiated hosts for stem cell assays will provide a first assessment of cell intrinsic vs. environmental factors in the formation of all blood cell lineages in normal as well as pathological conditions such as leukemia. In addition, these studies should shed light on parameters involved in bone marrow transplantation therapies. In the first strategy, purified retrovirally-marked stem cells will be engrafted into normal and mutant fetal mice by transplacental injection. Such cells should subsequently colonize the bone environment in a normal fashion and at the correct developmental time and contribute to hematopoiesis throughout adult life. A second strategy will combine germ- line transgenesis with somatic retroviral-mediated gene transfer to generate lines of mice that will introduce random stable proviral markers into hematopoietic stem cells as they develop in the normal environment. The key principal is the germ-line introduction of chimeric retroviral transgenes whose expression is targeted to cells comprising the niches within which hematopoietic stem cells proliferate. This will result in local production of marker virus which will infect the stem cells, inactivate itself local production of marker virus which will infect he stem cells, inactivate itself and therefore uniquely and permanently mark the cells and their resultant clonal progeny. The expression of marker virus is designed to occur only in the temporal frame during which stem cells first appear and clonally expand during ontogeny. Sequential and quantitative Southern blot analysis of proviral marker distribution in peripheral blood cell types obtained from animals derived from both approaches will define the developmental and proliferative behavior of the stem cell in its normal environment. The second proposed set of experiments will provide inroads to a precise molecular dissection of regulatory mechanisms which function in proliferation/differentiation decisions of primitive stem cells. Strategies are proposed to analyze gene expression directly in purified stem cells and to isolate stem cell specific members of regulatory gene families. These will provide direct insight into hematopoietic regulation as it functions in normal blood formation and malfunction to yield cancerous or other hematologic disorders.
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1995 — 2009 |
Lemischka, Ihor R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Molecular Studies of Hematopoietic Stem Cells
Hematopoiesis is a complex hierarchically-organized developmental system where progressive states are governed by properly balanced cell-fate decisions. The outcome of these processes is the continuous and lifelong production of at least eight distinct lineages of mature blood cells. The origin of the hematopoietic hierarchy lies in a rare population of self-renewing multipotent stem cells. Much biological and physical information has been obtained which collectively describes hematopoietic stem cells. In contrast, very little is known about the molecular biology of these cells. There have been few insights into the components and overall nature of the regulatory machinery which orchestrates the correct balance of self-renewal vs. commitment,: as well as other important developmental choices. A molecular understanding of stem cell regulation will provide a new context within which to view a normal or pathological hematopoietic process. In this proposal a range of existing and newly-emerging molecular technologies are focused on the biology of murine hematopoietic stem cells. In Aim One the gene-expression patterns which define the stem cell will be analyzed and will provide a first global stem cell molecular phenotype or "genetic space." To rationally explore the molecular phenotype, powerful bioinforrnatic tools will be applied. A major goal is to identify and characterize all gene-products whose expression segregates to the primitive stem/progenitor cell hierarchy. In Aim Two novel microarray approaches will be employed used to monitor the expression fluctuations in the stem cell "genetic space" as a function of differences in well‑defined biological properties. In all of these studies an emphasis is placed on molecular correlations with quantitatively measured functional activities of the stem cells rather than with their physical properties. Strategies will be developed to measure fluctuations as the stem/progenitor cell hierarchy develops in time. In Aim Three the biological roles of three novel cell-surface molecules isolated from stem cells will be defined using immunochemical and genetic gain and loss- of-function approaches. Collectively, the proposed studies will lay a necessary and firm foundation upon which to unravel stem cell regulation. The conserved properties of the murine and human hematopoietic systems suggest that the proposed studies could have a direct impact on human stem cell transplantation, ex vivo expansion, gene therapy and the etiology of leukemias as well as other disorders.
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2007 — 2010 |
Lemischka, Ihor R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Global and Systems Level Analyses of Cell Fate Regulation in Mouse Es Cells @ Mount Sinai School of Medicine of Nyu
[unreadable] DESCRIPTION (provided by applicant): Abstract: Embryonic stem (ES) cell research holds great promise for the future of medicine. In particular, new insights into human development, the etiology of complex diseases, as well as novel therapies will emerge. Fulfillment of these promises depends on comprehensive understanding of the regulatory processes that control cell-fate decisions in ES cells. Many years of work with mouse ES cells have defined a number of regulatory molecules and signaling pathways that are required for self-renewal, and lineage-specific differentiation. However, much remains to be learned. Comparisons of mouse and human ES cells have revealed similarities, as well as differences. We have developed novel approaches for global functional genomic analyses of cell fate regulation in mouse ES cells. Here, we will apply these approaches to define a comprehensive functional "parts list" of molecules responsible for control of cell-fate decisions. Our strategies are based on short hairpin RNA-mediated loss-of-function analyses of candidate regulatory gene- products. Perturbation of individual molecules allows multi-level analyses of cell fate changes, as they occur in time. We will apply a systems biology strategy an analyze cell-fate control simultaneously at the epigenetic, transcriptional, and proteomic levels. Coupled with genome-wide technologies, such perturbations also allow provisional assembly of the "parts" into transcriptional, as well as other types of regulatory networks. Our major focus will be on the control of ES cell self-renewal. Subsequently, we will initiate efforts to dissect the mechanisms that control commitment to specific cell lineages. Our results will provide a firm foundation for understanding ES cell fate regulation in a deep, systems level manner. They will also provide a framework and the tools for comprehensive analyses of cell-fate regulation in the human ES cell system. Ultimately, our studies will also provide reliable and robust avenues to direct ES cell differentiation to specific cell types and populations. [unreadable] [unreadable] [unreadable]
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2010 |
Lemischka, Ihor R |
RC1Activity Code Description: NIH Challenge Grants in Health and Science Research |
Pursuing Pluripotency @ Icahn School of Medicine At Mount Sinai
DESCRIPTION (provided by applicant): Human induced pluripotent cells (hiPSC) hole enormous promise for regenerative medicine. We propose to explore new methodologies to generate hiPSC without the use of transgene vectors and to develop ways to understand the kinetics, biochemistry and molecular biology of the reprogramming process. In the first part, we have built systems where individual or combinations of reprogramming factors can be controlled artificially and robustly. This will allow us to address questions regarding the temporal sequence of factor requirement, the duration of factor expression necessary for successful reprogramming and other issues. This system will also set the stage for both broad and focused chemical genomics approaches to identify chemical compound that replace individual or combinations of reprogramming factors. The second part builds of results from the first and will explore the dynamics of reprogramming at the epigenetic, transcriptional, steady state mRNA and proteomic levels. This will provide key mechanistic insights into how reprogramming occurs and into the very nature of the pluripotent state. PUBLIC HEALTH RELEVANCE: The proposed studies will provide a better understanding of the reprogramming process by which adult human cells can be converted to induced pluripotent cells (hiPSC). These cells can be derived from patients and will provide important insights into the etiology of complex diseases and potential cell replacement therapies in the future. Other proposed studies will provide more efficient and safer, clinically applicable approaches to generate hiPSC.
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2012 — 2015 |
Lemischka, Ihor R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Global & Systems Level Analyses of Cell Fate Regulation @ Icahn School of Medicine At Mount Sinai
DESCRIPTION (provided by applicant): Pluripotent stem cells hold enormous promise for biomedicine. Indeed the recent development of induced pluripotent stem cell (iPSC) procedures allows the generation of patient-specific pluripotent cells from any individual. Induced pluripoten stem cells closely resemble their embryo-derived counterparts, embryonic stem cells (ESC). Currently, however, the exact degree of identity between ESC and iPSC is unclear. Human (h) iPSC are genetically identical to the person of origin and thus, carry the exact complement of genetic variables that cause or predispose the patient to disease. This enables the development of exciting and unprecedented avenues to model disease etiology, better diagnostic and pharmaceutical reagents and in time, defined mature cell populations suitable for transplantation-based therapies. In order to realize the potential of pluripotent cells a detailed, quantitative understanding of their regulatory mechanisms is critical. By their nature, complex biological systems cannot be understood using reductionist approaches. Rather, systems biology paradigms that merge global experimental technologies with computational approaches are required to reveal how a cell processes biological information to affect a change in fate. Together with our computational colleagues we have developed such a systems approach and applied it to study the regulation of mouse (m) ESC, the founding and best-characterized member of the pluripotency pantheon. In the current proposal we will extend our studies to define how biological information is processed by mESC over time after a defined perturbation. We will focus on transcriptional regulators such as Nanog, Esrrb and Tbx3, and others that are necessary for pluripotency and measure their functions at the epigenetic, transcriptional, mRNA, microRNA and proteomic levels. The interactions among these factors in regulatory modules will also be explored. These studies will provide an unprecedented dynamic view of ESC regulation. In a sense, how individual pluripotent cells traverse the Waddington Landscape will be measured and visualized. Other genetic loss-of-function studies will be pursued to identify the complete panel of protein-coding gene-products that together function to maintain the pluripotent state and its transitions. We will also apply novel analytical tools to identify molecules that alter ESC properties in subtle and previously undefinable ways. Selected candidate molecules have already emerged and will be studies in detail. Finally, the existence of multiple alternative pluripotency regulatory network configuration will be explored and the roles of biological noise as well as stochasticity in ESC regulation will be addressed.
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2013 — 2014 |
Gelb, Bruce D [⬀] Lemischka, Ihor R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Human Induced Pluripotent Cell Models of Pediatric Cardiac Disorders @ Icahn School of Medicine At Mount Sinai
DESCRIPTION (provided by applicant): A long-standing barrier in cardiovascular biology research has been the inability to maintain human cardiomyocytes in long-term cell culture. Since 2007 when the possibility of reprogramming terminally differentiated human cells like skin fibroblasts into pluripotent stem cells was demonstrated, it became possible to generate human cardiomyocytes in vitro, enabling the study of human primary myocardial diseases. For this project, we intend to study inherited forms of childhood myocardial disease: hypertrophic cardiomyopathy (HCM) associated with RAS signaling abnormalities and atrial muscle-related tachycardia associated with increased HRAS signaling. The RASopathies are a family of autosomal dominant disorders caused by missense mutations in genes encoding RAS/MAPK pathway proteins. HCM is common in the RASopathies and multifocal atrial tachycardia (MAT) is specifically observed in one disorder, Costello syndrome, which is caused by gain-of-function HRAS mutations. For SPECIFIC AIM 1, the PIs hypothesize that RASopathy- associated HCM arises through signaling pathway activation that differs among the specific disorders. To test this, they will use existing human iPSC lines for two RASopathies that exhibit cardiomyocyte hypertrophy. Isolated ventricular cardiomyocytes harboring LEOPARD and cardiofaciocutaneous syndrome-causing mutations will be characterized with respect to signal transduction, intracellular calcium handling and contractility among cells. For SPECIFIC AIM 2, the PIs hypothesize that MAT in Costello syndrome is caused by perturbations in intracellular calcium handling induced by altered signaling from HRAS. To test this, atrial cardiomyocytes, differentiated from Costello syndrome iPSC lines, will be assessed for Ca2+ transients, electrophysiology and response to relevant anti-arrhythmic drugs. For SPECIFIC AIM 3, the PIs hypothesize that RASopathy-associated HCM will be reversed by inhibitors that address mutation-specific signaling perturbations. To study this, iPSC-derived LEOPARD and cardiofaciocutaneous syndrome cardiomyocytes will be treated with signaling pathway inhibitors to reverse hypertrophy. Dose response curves will be established. Combination therapies will be tested for efficacy at lower doses. Effects of therapies on the autophagy defect in LEOPARD syndrome cardiomyocytes will be determined. Broadly, these studies will harness the power of the new iPSC technology to elucidate the pathogenesis of myocardial disease associated with RASopathies: HCM and atrial arrhythmias. These studies could have important impact on the development of novel therapeutic strategies for these myocardial diseases, for which our current approaches are not curative.
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2014 — 2017 |
Drew, Patrick James Jabs, Ethylin Wang Kraft, Reuben H. Lemischka, Ihor R Peter, Inga Richtsmeier, Joan Therese Romitti, Paul A (co-PI) [⬀] Schadt, Eric E (co-PI) [⬀] |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Craniosynostosis Network @ Icahn School of Medicine At Mount Sinai
? DESCRIPTION (provided by applicant): The long-term goal of the Program Project, Craniosynostosis Network, is to elucidate normal and abnormal craniofacial biology to ultimately improve the treatment of craniofacial disorders. Craniosynostosis (CS) and other skull abnormalities are among the most common human malformations and usually require surgical and medical intervention. Our Network will integrate the efforts of scientists with diverse expertise including anthropology, morphometry, imaging, birth defects, developmental biology, genetics, genomics, epidemiology, statistics, & system biology to explore the determinants of the fate of the relevant mesenchymal progenitor cells, and how abnormalities in the processes of osteogenesis contribute to disorders such as global skull growth abnormality, premature closure of sutures, in particular the coronal suture. We will use humans and mouse model systems to study normal development and malformations that characterize birth defects such as Apert, Crouzon, and Muenke syndromes & coronal nonsyndromic craniosynostosis. Our research design will be multidisciplinary including imaging, genomics, computational modeling & stem cell research; and evolutionary, developmental, & systems biology. Our approach will be hypothesis and discovery-driven, and we will generate and integrate a wide variety of human genomic, imaging, & laboratory data. The Network will be based at Mount Sinai Medical Center with the contact Principal Investigator (PI), Ethylin Wang Jabs, and multiple PIs, Inga Peter, Eric Schadt and Ihor Lemischka, and at Pennsylvania State University with MPIs Joan Richtsmeier, Patrick Drew, and Reuben Kraft. Our international and national collaborating institutions include: Hospital Necker-Enfants Maladies (France), University Hospital Heidelberg (Germany), and Hospital Sant Joan de Deu (Spain); Oxford University (UK), the International Craniosynostosis Consortium at University of California at Davis; New York State birth defect registry involved with the National Birth Defects Prevention Study based at Univ. of Iowa, directed by MPI Paul Romitti; New York University, Pennsylvania State Milton S. Hershey Medical Center; Boston Children's Hospital; Yale University, Univ. of Texas at Southwestern, and Johns Hopkins University. Our Advisory Committee includes experts in developmental biology, genomics, and system biology: Philippe Soriano from Mount Sinai Medical Center, James Sharpe of the Centre for Genomic Regulation, Barcelona, Spain, Alec Wilson of NIH NHGRI, & Richard Bonneau of New York University. Our proposal consists of Project I From Skull Shape to Cell Activity in Coronal Craniosynostosis, Project II Genomics Approaches to Coronal Nonsyndromic Craniosynostosis, & Project III Systems Biology of Bone in Coronal Nonsyndromic Craniosynostosis; and two Cores: Administrative Core A and Molecular/Analytic Core B. The investigators, by engaging as an integrated group in the study of complex biological networks, and by utilizing innovative and state-of-the-art technologies, will foster an outstanding research environment. The Network is strongly committed to sharing & disseminating our findings to the scientific community at large.
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2014 — 2017 |
Lemischka, Ihor R Moore, Kateri A. [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Programming Hematopoiesis @ Icahn School of Medicine At Mount Sinai
DESCRIPTION (provided by applicant): Hematopoietic stem cell (HSC) transplantation is widely used to treat a variety of disorders. Despite advances in the use of cord blood and mobilized stem cells, donor material remains limited. This is due to limited stem cells in cord blood, poor mobilization, and the lack of ethnic diversity to provide sufficient matched material. Despite intensive efforts there has been very limited success in generating transplantable HSCs from embryonic stem cells or more recently, from induced pluripotent stem cells. We asked if HSCs could be directly programmed from murine fibroblasts with defined combinations of transcription factors (TFs). A set of 18 candidate TFs was identified and introduced into mouse embryonic fibroblasts (MEFs) containing a reporter with expression specific to primitive endothelial and hematopoietic stem/progenitor cells. Transduction with the 18 TF cocktail resulted in activation of the reporter. Sequential experiments evaluated the requirement of each TF. We identified a set of 4 TFs (Gata2, Gfi1b, cFos, and Etv6) as sufficient for efficient hemogenic induction. These 4 TFs induce a dynamic, multi-stage process that progresses through an endothelial-like intermediate. Transduced MEFs first generate cells with a global endothelial-like gene expression program that are organized into characteristic structures. We show that a Reporter+Sca1+Prominin1+ precursor emerges with hematopoietic activity. Emergent hematopoietic cells have a global gene expression program highly similar to bona fide HSCs most specifically to the specifying HSCs from Aorta Gonad Mesonephros (AGM), placenta, and early fetal liver. We also detect a subpopulation of cells with an HSC cell surface phenotype. Upon transfer of the 4 TFs to inducible vectors where expression can be turned off and the use of aggregation cultures we have been able to demonstrate the development of multi-lineage in vitro colonies. Collectively, our results strongly suggest that the 4 TFs recapitulate a complex developmental program highly similar to fetal hemogenesis. These data are now published in Cell Stem Cell. In this proposal we will further our initial studies with inducible hemogenic programming systems. We will then identify the kinetics of the hemogenic process with a focus on defining the exact temporal requirement for exogenous TF expression. We will employ a broad range of in vitro and in vivo assays. We also propose an in-depth molecular analysis of our hemogenic system emphasizing the generation of intermediate endothelial-like cells and the ultimate emergence of HSC-like populations. The molecular analyses will focus on global mRNA and microRNA expression profiling. Finally, we will build on our preliminary results in the human system. The human studies will closely follow our efforts in the mouse. Collectively, our studies will rigorously establish whether a hemogenic program that yields fully functional HSCs can be specified in vitro and provide a future source of material for regenerative medicine.
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