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Mickie Bhatia

Publications and source records attributed to Mickie Bhatia.

At least 19 recordsLinked to original sources
PubMed2006

Hedgehog modulates cell cycle regulators in stem cells to control hematopoietic regeneration.

The signals that control the regenerative ability of hematopoietic stem cells (HSCs) in response to damage are unknown. Here, we demonstrate that downstream activation of the Hedgehog (Hh) signaling pathway induces cycling and expansion of primitive bone marrow hematopoietic cells under homeostatic conditions and during acute regeneration. However, this effect is at the expense of HSC function, because continued Hh activation during regeneration represses expression of specific cell cycle regulators, leading to HSC exhaustion. In vivo treatment with an inhibitor of the Hh pathway rescues these transcriptional and functional defects in HSCs. Our study establishes Hh signaling as a regulator of the HSC cell cycle machinery that balances hematopoietic homeostasis and regeneration in vivo.

Animals↗
PubMed2006

Clonal isolation of hESCs reveals heterogeneity within the pluripotent stem cell compartment.

Human embryonic stem cell (hESC) lines are known to be morphologically and phenotypically heterogeneous. The functional nature and relationship of cells residing within hESC cultures, however, has not been evaluated because isolation of single hESCs is limited to drug or manual selection. Here we provide a quantitative method using flow cytometry to isolate and clonally expand hESCs based on undifferentiated markers, alone or in combination with a fluorescent reporter. This method allowed for isolation of stage-specific embryonic antigen-3-positive (SSEA-3+) and SSEA-3- cells from hESC cultures. Although both SSEA-3+ and SSEA-3- cells could initiate pluripotent hESC cultures, we show that they possess distinct cell-cycle properties, clonogenic capacity and expression of ESC transcription factors. Our study provides formal evidence for heterogeneity among self-renewing pluripotent hESCs, illustrating that this isolation technique will be instrumental in further dissecting the biology of hESC lines.

Antigens, Tumor-Associated, Carbohydrate↗
PubMed2006

Derivation and characterization of hematopoietic cells from human embryonic stem cells.

In vitro, the aggregation of pluripotent human embryonic stem cells (hESC) into cell clusters termed embryoid bodies (EB) allows for the spontaneous differentiation of hESC into progeny representing endoderm, mesoderm, and ectoderm lineages. During human EB (hEB) differentiation, stochastic emergence of hematopoietic cells can be enhanced by a combination of hematopoietic cytokines and the ventral mesoderm inducer bone morphogenetic protein (BMP)-4. Dependent on the presence of hematopoietic cytokines and BMP-4, vascular endothelial growth factor (VEGF-A165) selectively promotes erythropoietic development toward the primitive lineage. The effects of VEGF-A165 can be augmented by erythropoietin (EPO). Hematopoietic cells are derived from a rare subpopulation of hemogenic precursors during hEB development. These hemogenic precursors lack CD45, but express PECAM-1, Flk-1, and VE-cadherin (hereinafter CD45(neg)PFV) and are solely responsible for hematopoietic cell fate. Human ESC-derived hematopoietic cells have similar colony and cellular morphologies to those derived from committed adult hematopoietic tissues, and also show repopulating capacity in immune deficient mice after intrabone marrow transplantation. In this chapter, we describe methods that have been successfully applied in our laboratory, including (1) generation of hematopoietic cells by EB formation; (2) augmentation of hematopoiesis by use of hematopoietic cytokines and BMP-4; (3) promotion of erythropoietic development by addition of VEGF-A165 and EPO; (4) isolation of CD45(neg)PFV hemogenic precursors and generation of hematopoietic cells from these precursors; and (5) characterization of hESC-derived hematopoietic cells in vitro and in vivo.

Animals↗
PubMed2006

Retroviral transduction of hematopoietic progenitors derived from human embryonic stem cells.

It has been recently identified that cytokines and BMP-4 promote hematopoiesis from human embryonic stem cells (hESC) and that, before hematopoietic commitment, a rare subpopulation of cells lacking CD45, but expressing PECAM-1, Flk-1, and VE-cadherin (hereinafter termed CD45(neg)PFV precursors), are exclusively responsible for hematopoietic cell fate on cytokine stimulation. Efficient strategies to stably transduce these hematopoietic precursors specifically generated from hESCs would provide a novel and desirable tool to study hematopoietic development through the introduction and characterization of candidate genes suspected to regulate self-renewal processes of hESC-derived hematopoietic cells or dynamically track hESC-derived hematopoietic stem cells in vivo. To date, only transient transfection and stable transduction using lentiviral vectors have been reported in undifferentiated hESC followed by random and spontaneous differentiation into different cell types. However, protocols for stable transduction of hematopoietic progenitors prospectively derived from hESC need to be developed yet. In the present chapter, we described detailed methods on the recently characterized and optimized GALV-pseudotyped retroviral gene transfer strategy to stably transduce the hematopoietic progenitor cells prospectively derived from CD45(neg)PFV hemogenic precursors as a vital tool to study hematopoietic development and to characterize candidate genes suspected to eventually confer robust and sustained repopulating ability to hESC-derived hematopoietic cells.

Antigens, CD↗
PubMed2005

Glycogen synthase kinase-3 is an in vivo regulator of hematopoietic stem cell repopulation.

The in vivo regulation of hematopoietic stem cell (HSC) function is poorly understood. Here, we show that hematopoietic repopulation can be augmented by administration of a glycogen synthase kinase-3 (GSK-3) inhibitor to recipient mice transplanted with mouse or human HSCs. GSK-3 inhibitor treatment improved neutrophil and megakaryocyte recovery, recipient survival and resulted in enhanced sustained long-term repopulation. The output of primitive Lin(-)c-Kit(+)Sca-1(+) cells and progenitors from HSCs increased upon GSK-3 inhibitor treatment without altering secondary repopulating ability, suggesting that the HSC pool is maintained while overall hematopoietic reconstitution is increased. GSK-3 inhibitors were found to modulate gene targets of Wnt, Hedgehog and Notch pathways in cells comprising the primitive hematopoietic compartment without affecting mature cells. Our study establishes GSK-3 as a specific in vivo modulator of HSC activity, and suggests that administration of GSK-3 inhibitors may provide a clinical means to directly enhance the repopulating capacity of transplanted HSCs.

Animals↗
PubMed2005

A unique population of bone marrow cells migrates to skeletal muscle via hepatocyte growth factor/c-met axis.

Cells expressing the CD45-associated hematopoietic marker are predominantly present in the mammalian bone marrow (BM), but have recently been shown to also reside in the skeletal muscle and potentially participate in muscle repair. Despite the consistent observations, the specific relationship and potential migration of CD45+ cells in the BM versus CD45+ cells residing in the muscle remain unclear, in addition to any understanding of the factors that may regulate the trafficking of CD45+-derived BM cells to skeletal muscle upon i.v. transplantation. Here, transplantation of BM-derived cells fully replaced the CD45+ fraction of skeletal muscle, but gave rise to progenitor cells with distinct hematopoietic lineage capacity from CD45+ cells residing in the BM. Using transwell migration assays, a subset of BM cells was shown to migrate exclusively to mature skeletal muscle cells and not BM-derived stromal cells. Unlike migration of BM cells to stroma, myofiber induced migration of BM-derived cells was not affected by stromal-derived factor-1 (SDF-1) neutralization or CXCR4-blocking antibody, but could be reduced by addition of c-met-blocking antibody and augmented by hepatocyte growth factor (HGF), the putative ligand for c-met. We suggest that the BM compartment consists of a functionally complex population of CD45+ progenitors that includes a subset of HGF/c-met responsive cells capable of migration to skeletal muscle. This previously unappreciated basis for cellular tracking now aids in defining regulatory networks that distinguish the stem cell niche of the BM versus skeletal muscle microenvironments.

Animals↗
PubMed2005

Generation of hematopoietic repopulating cells from human embryonic stem cells independent of ectopic HOXB4 expression.

Despite the need for alternative sources of human hematopoietic stem cells (HSCs), the functional capacity of hematopoietic cells generated from human embryonic stem cells (hESCs) has yet to be evaluated and compared with adult sources. Here, we report that somatic and hESC-derived hematopoietic cells have similar phenotype and in vitro clonogenic progenitor activity. However, in contrast with somatic cells, hESC-derived hematopoietic cells failed to reconstitute intravenously transplanted recipient mice because of cellular aggregation causing fatal emboli formation. Direct femoral injection allowed recipient survival and resulted in multilineage hematopoietic repopulation, providing direct evidence of HSC function. However, hESC-derived HSCs had limited proliferative and migratory capacity compared with somatic HSCs that correlated with a distinct gene expression pattern of hESC-derived hematopoietic cells that included homeobox (HOX) A and B gene clusters. Ectopic expression of HOXB4 had no effect on repopulating capacity of hESC-derived cells. We suggest that limitations in the ability of hESC-derived HSCs to activate a molecular program similar to somatic HSCs may contribute to their atypical in vivo behavior. Our study demonstrates that HSCs can be derived from hESCs and provides an in vivo system and molecular foundation to evaluate strategies for the generation of clinically transplantable HSC from hESC lines.

Animals↗
PubMed2005

Human embryonic stem cells maintained in the absence of mouse embryonic fibroblasts or conditioned media are capable of hematopoietic development.

To date, hematopoietic development of human embryonic stem cells (hESCs) has been limited to cell lines cultured in the presence of either mouse embryonic fibroblasts (MEFs) or MEF-conditioned media (MEF-CM). Anonymous xenogenic factors from MEFs or MEF-CM complicate studies of hESC self-renewal and also raise concerns for the potential clinical applications of generating primitive hematopoietic cells from hESC lines maintained under these ambiguous conditions. Here, we demonstrate that hESCs can be cultured over 30 passages in defined conditions in the absence of MEFs or MEF-CM using only serum replacement (SR) media and high concentrations of basic fibroblast growth factor (SR-bFGF). Similar to hESCs cultured in MEF-CM, hESCs cultured in SR-bFGF sustained characteristics of undifferentiated hESCs, proliferative potential, normal karyotype, in vitro and in vivo 3 germ-layer specification and gave rise to hemogenic-endothelial precursors required for subsequent primitive hematopoietic development. Our report demonstrates that anonymous factors produced by feeder cells are not necessary for hESC maintenance and subsequent hematopoietic specification, thereby providing a defined system for studies of hESC self-renewal and hESC-derived hematopoiesis.

Alkaline Phosphatase↗
PubMed2005

Hierarchical and ontogenic positions serve to define the molecular basis of human hematopoietic stem cell behavior.

The molecular basis governing functional behavior of human hematopoietic stem cells (HSCs) is largely unknown. Here, using in vitro and in vivo assays, we isolate and define progenitors versus repopulating HSCs from multiple stages of human development for global gene expression profiling. Accounting for both the hierarchical relationship between repopulating cells and their progenitors, and the enhanced HSC function unique to early stages of ontogeny, the human homologs of Hairy Enhancer of Split-1 (HES-1) and Hepatocyte Leukemia Factor (HLF) were identified as candidate regulators of HSCs. Transgenic human hematopoietic cells expressing HES-1 or HLF demonstrated enhanced in vivo reconstitution ability that correlated to increased cycling frequency and inhibition of apoptosis, respectively. Our report identifies regulatory factors involved in HSC function that elicit their effect through independent systems, suggesting that a unique orchestration of pathways fundamental to all human cells is capable of controlling stem cell behavior.

Animals↗
PubMed2005

Hematopoietic development from human embryonic stem cell lines.

The most common human cell-based therapy applied today is hematopoietic stem cell (HSC) transplantation. Currently, human bone marrow, mobilized peripheral blood, and umbilical cord blood represent the major sources of transplantable HSCs, but their availability for use is limited by both compatibility between donor and recipient and required quantity. Although increasing evidence suggests that somatic HSCs can be expanded to meet current needs, their in vivo potential is concomitantly compromised after ex vivo culture. In contrast, human embryonic stem cells (hESC) possess indefinite proliferative capacity in vitro and have been shown to differentiate into the hematopoietic cell fate, giving rise to erythroid, myeloid, and lymphoid lineages using a variety of differentiation procedures. Human ESC-derived hematopoietic cells emerge from a subset of embryonic endothelium expressing PECAM-1, Flk-1, and VE-Cadherin, but lacking CD45 (CD45negPFV). These CD45negPFV precursors are exclusively responsible for hematopoietic potential of differentiated hESCs. hESC-derived hematopoietic cells show similar clonogenic capacity and primitive phenotype to somatic sources of hematopoietic progenitors and possess limited in vivo repopulating capacity in immunodeficient mice, suggestive of HSC function. Here, we will review current progress in studies of hESC-derived hematopoietic cells and discuss the potential precincts and applications.

Cell Culture Techniques↗
PubMed2005

Intra-bone marrow transplantation facilitates pauci-clonal human hematopoietic repopulation of NOD/SCID/beta2m(-/-) mice.

OBJECTIVE: Intra-bone marrow transplantation (IBMT) has been shown to improve the limit of detection of primitive human SCID-repopulating cells (SRC) in NOD/SCID mice when compared to intravenous transplantation. We sought to further refine detection of SRC by comparing NOD/SCID mice to the more sensitive NOD/SCID/beta2m(-/-)strain as IBMT recipients of limiting numbers of purified primitive human hematopoietic cells. MATERIALS AND METHODS: Purified human Lin(-)CD34(+)CD38- cells at limiting doses were delivered by IBMT into NOD/SCID and NOD/SCID/beta2m(-/-) strains of recipient mice. Six weeks posttransplantation, injected and noninjected bones were analyzed separately for multilineage human hematopoietic chimerism. RESULTS: NOD/SCID/beta2m(-/-) mice are superior recipients for IBMT and show a trend toward increased levels of human hematopoietic engraftment. In addition, in contrast to NOD/SCID recipients, NOD/SCID/beta2m(-/-) mice were reconstituted with as few as five highly purified cells, indicative of pauci-clonal repopulation. Analysis of injected and noninjected bones demonstrated that engrafting cells were capable of in vivo migration and expansion. Although SRC hematopoietic reconstitution of NOD/SCID mice is commonly lymphoid-dominant, multilineage analysis of separate bone sites following IBMT of purified cells revealed that a subset of mice was repopulated with a myeloid-dominant graft in at least one bone site, revealing that SRC are developmentally heterogeneous among Lin(-)CD34(+)CD38- cells and capable of distinct differentiation potential. CONCLUSION: IBMT into NOD/SCID/beta2m(-/-) mice provides a highly sensitive experimental transplantation assay for the detection of human hematopoietic repopulating cells and demonstrates that Lin(-)CD34(+)CD38- cells are more highly enriched for human SRC than originally predicted.

Animals↗
PubMed2005

Analysis of the human fetal liver hematopoietic microenvironment.

In the adult, hematopoietic stem cells (HSCs) are resident in the bone marrow (BM) compartment and are in direct association with the BM stromal microenvironment. However, human adult HSCs are largely quiescent and undergo limited self-renewal. This is in contrast to the higher frequency of cycling HSCs undergoing self-renewal during fetal development when hematopoiesis is transiently localized to the fetal liver (FL), suggesting that FL provides a more conducive microenvironment to support HSCs. Here, we provide phenotypic and molecular characterization of primary human FL stromal cells capable of supporting human repopulating progenitors. Qualitative and quantitative analysis revealed several properties unique to FL stromal cells compared to adult BM-derived stroma that included a greater than 10-fold enhanced proliferative capacity of FL stromal vs adult BM, and a 2-fold increase in the number of N-cadherin- and osteopontin-expressing cells. Supportive of extrinsic influences likely to modulate HSC expansion, global gene expression microarray analysis revealed that FL stroma has higher expression of regulators of the Wnt signaling pathway compared to adult BM stroma, which demonstrated an increased expression of the Notch signaling pathway. Our results suggest that human FL stromal cells provide a unique microenvironment to HSCs compared to adult BM stroma by controlling Wnt signaling of HSCs during human fetal hematopoietic development, while Notch signaling is tightly regulated by the HSC microenvironment in the adult. We propose that the human HSC niche is ontogenically controlled during human development to provide appropriate expansion of fetal HSCs and subsequent maintenance of adult HSCs.

Adult↗
PubMed2005

Derivation of the hematopoietic stem cell compartment from human embryonic stem cell lines.

Since the derivation of human embryonic stem cell (hESC) lines, both experimental and clinical hematology have generated rising interest in differentiating functional hematopoietic progenitors from these pluripotent cell lines. Although the ability to generate primitive hematopoietic cells with in vivo reconstitution function has direct clinical application, success of this approach awaits a more in-depth understanding of factor and mechanisms that govern specification of pluripotent hESCs to hematopoietic lineage specification. Here, we highlight some of our laboratory's recent efforts in this area, in the hopes of providing insights toward this complex yet important area of biomedical research.

Cell Line↗
PubMed2005

Human embryonic stem cells: potential tool for achieving immunotolerance?

The derivation of human embryonic stem cells (hESCs), whose in vitro differentiation might be directed toward different cell types, has raised the hope for cell replacement therapies. Despite the emerging reports to differentiate hESCs into specific lineages and then to distinct mature cell subsets, there are still several issues that need to be resolved before transplantation of these cells can be realized. In this context, immune rejection by the host immune system has been considered to be one of the greatest hurdles for cellular transplantation. However, recent data support the concept that hESCs and/or their differentiated derivatives possess immune-privileged properties, suggesting that cells derived from hESC may provide a potential tool for induction of immunetolerance. Currently, our understanding of the tolerogenic potential of hESCs is limited to assessment by in vitro assays or xenogenic transplantation approaches in vivo. Human ESCs express low levels of major histocompatability complex (MHC)-I antigens and lack expression of MHC-II antigens and costimulatory molecules, and are not recognized by natural killer cells and inhibit T-cell induced-stimulation by third-party antigen-presenting cells. Upon injection into immunocompetent mice, hESCs are unable to induce an immune response as demonstrated by their inability to induce an inflammatory response. Based on these initial observations, further studies in hESCs immunobiology are warranted and may reveal unique mechanisms that account for the immunological properties of hESCs. Here, we explore the prospect of using hESCs and their derivatives for immunomodulation and tolerance induction.

Animals↗
PubMed2005

Genetic manipulation of human embryonic stem cells: a system to study early human development and potential therapeutic applications.

The successful derivation of human embryonic stem cell (hESC) lines by Thomson and colleagues [Thomson et al., 1998] provided a new area of investigation in both regenerative medicine and early human development. Fundamental study of the molecular and cellular mechanisms responsible for normal lineage development will rely on reproducible protocols to direct the differentiation of hESCs into specific lineages of interest and genetically manipulate both hESCs and their derivatives. Identifying standards for maintenance of hESCs, methods for controlled differentiation and genetic manipulation of hESCs and their derivatives will provide a foundation to explore their potential therapeutic use in cell and gene therapy. In the present review, our goal is to outline the latest advances in the field with particular focus on how hESCs and their derivatives can be genetically altered, how this may be useful in better understanding the cellular and molecular events of lineage differentiation, and how deregulation of these cellular processes may lead to abnormal development and disease.

Cell Differentiation↗
PubMed2005

Identification of growth factor conditions that reduce ex vivo cord blood progenitor expansion but do not alter human repopulating cell function in vivo.

BACKGROUND AND OBJECTIVES: Ex vivo expansion of primitive hematopoietic cells for transplantation is an important step to realizing the optimal clinical potential of human cord blood (CB). We aimed to characterize minimal growth factor (GF) conditions that allow ex vivo expansion of primitive cells, including candidate hematopoietic stem cells. DESIGN AND METHODS: Here, we directly investigated the effect of thrombopoietin (TPO) on progenitors and repopulating cells using serum-free culture of CB Lin-CD34+CD38- cells in two different minimal GF conditions: stem cell factor (SCF)+FLT-3-L (termed S/F) and SCF+FLT-3-L+TPO (termed S/F/T). RESULTS: While S/F media supported only low levels of total cell and CFU (colony-forming unit) expansion, the addition of TPO (S/F/T) partially restored cell proliferation, and completely restored CFU expansion to levels observed using full GF conditions (SCF+FLT-3-L+interleukin (IL)-3 (IL-3)+ IL-6+ granulocyte colony-stimulating factor (G-CSF). Intravenous transplantation of either S/F- or S/F/T-expanded cells into NOD/SCID mice resulted in similar frequencies and levels of multilineage reconstitution. INTERPRETATION AND CONCLUSIONS: The use of minimal cytokine stimulation and simultaneous assessment of CFU and SRC indicate that hematopoietic progenitors and in vivo-detected repopulating cells are differentially responsive to TPO; CFU expand in response to TPO but SRC do not. In addition, our study suggests that TPO can functionally replace IL-3+IL-6+G-CSF for CFU expansion of ex vivo cultured CB Lin-CD34+CD38- hematopoietic stem cells.

ADP-ribosyl Cyclase 1↗