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In JoVE (1)
Other Publications (15)
- Proceedings of the National Academy of Sciences of the United States of America
- Experimental Hematology
- Experimental Hematology
- Methods in Molecular Biology (Clifton, N.J.)
- Leukemia Research
- Stem Cells (Dayton, Ohio)
- Annals of the New York Academy of Sciences
- Current Opinion in Hematology
- Blood
- Cell Stem Cell
- Proceedings of the National Academy of Sciences of the United States of America
- Current Protocols in Stem Cell Biology
- Blood
- Nature
- Haematologica
Articles by Shannon McKinney-Freeman in JoVE
JoVE General
Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells
Childrens Hospital, Harvard Stem Cell Institute, Harvard Medical School
This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.
Other articles by Shannon McKinney-Freeman on PubMed
Muscle-derived Hematopoietic Stem Cells Are Hematopoietic in Origin
It has recently been shown that mononuclear cells from murine skeletal muscle contain the potential to repopulate all major peripheral blood lineages in lethally irradiated mice, but the origin of this activity is unknown. We have fractionated muscle cells on the basis of hematopoietic markers to show that the active population exclusively expresses the hematopoietic stem cell antigens Sca-1 and CD45. Muscle cells obtained from 6- to 8-week-old C57BL/6-CD45.1 mice and enriched for cells expressing Sca-1 and CD45 were able to generate hematopoietic but not myogenic colonies in vitro and repopulated multiple hematopoietic lineages of lethally irradiated C57BL/6-CD45.2 mice. These data show that muscle-derived hematopoietic stem cells are likely derived from the hematopoietic system and are a result not of transdifferentiation of myogenic stem cells but instead of the presence of substantial numbers of hematopoietic stem cells in the muscle. Although CD45-negative cells were highly myogenic in vitro and in vivo, CD45-positive muscle-derived cells displayed only very limited myogenic activity and only in vivo.
Altered Phenotype and Reduced Function of Muscle-derived Hematopoietic Stem Cells
Skeletal muscle-derived cells have the potential to repopulate the major peripheral blood lineages of lethally irradiated mice and thus behave like hematopoietic stem cells (HSC). We have recently shown that muscle cells with HSC activity (ms-HSC) express CD45 and Sca-1, suggesting a hematopoietic origin. Here we sought to clarify contradictions in the literature regarding the phenotype of ms-HSC and precisely define the hematopoietic origin of these cells.
Circulating Hematopoietic Stem Cells Do Not Efficiently Home to Bone Marrow During Homeostasis
Hematopoietic stem cells (HSC), normally resident in bone marrow, can be detected in the murine and human circulation. It is thought that HSC move in and out of bone marrow daily and that returning HSC are generally equivalent to their bone marrow counterparts in phenotype and function. However, large numbers of mononuclear blood cells are required to rescue animals from lethal irradiation, indicating either that the prevalence of circulating HSC is low, or they are inherently deficient in their repopulating ability. Accordingly, recent data suggest that circulating HSC may be unable to stably engraft WBM under homeostatic conditions. The purpose of this study was to explore these dynamics in detail using parabiosis and bone marrow transplantation.
Isolation and Characterization of Side Population Cells
The protocol for isolation of side population (SP) cells was originally established for murine bone marrow hematopoietic stem cells (HSCs), but it has also been adapted for other species and tissues. This purification strategy offers a simple and reproducible strategy to obtain a highly homogeneous population of HSCs. The method is based on the differential efflux of the fluorescent DNA-binding dye Hoechst 33342 from stem cells relative to nonstem cells. The protocols outlined in this chapter describe the isolation of murine SP cells from both bone marrow and skeletal muscle using the fluorescent DNA-binding dye Hoechst 33342. In these tissues, the SP cells that are isolated are HSCs.
Differential MRNA Processing in Hematopoietic Stem Cells
Hematopoietic stem cells (HSCs) maintain tissue homeostasis by rapidly responding to environmental changes. Although this function is well understood, the molecular mechanisms governing this characteristic are largely unknown. We used a sequenced-based strategy to explore the role of both transcriptional and post-transcriptional regulation in HSC biology. We characterized the gene expression differences between HSCs, both quiescent and proliferating, and their differentiated progeny. This analysis revealed a large fraction of sequence tags aligned to intronic sequences, which we showed were derived from unspliced transcripts. A comparison of the biological properties of the observed spliced versus unspliced transcripts in HSCs showed that the unspliced transcripts were enriched in genes involved in DNA binding and RNA processing. In addition, levels of unspliced message decreased in a transcript-specific fashion after HSC activation in vivo. This change in unspliced transcript level coordinated with increases in gene expression of splicing machinery components. Combined, these results suggest that post-transcriptional regulation is important in HSC activation in vivo.
The Cdx-hox Pathway in Hematopoietic Stem Cell Formation from Embryonic Stem Cells
Embryonic stem cells (ESCs) differentiated in vitro will yield a multitude of hematopoietic derivatives, yet progenitors displaying true stem cell activity remain difficult to obtain. Possible causes are a biased differentiation to primitive yolk sac-type hematopoiesis, and a variety of developmental or functional deficiencies. Recent studies in the zebrafish have identified the caudal homeobox transcription factors (cdx1/4) and posterior hox genes (hoxa9a, hoxb7a) as key regulators for blood formation during embryonic development. Activation of Cdx and Hox genes during the in vitro differentiation of mouse ESCs followed by co-culture on supportive stromal cells generates ESC-derived hematopoietic stem cells (HSCs) capable of multilineage repopulation of lethally irradiated adult mice. We show here that brief pulses of ectopic Cdx4 or HoxB4 expression are sufficient to enhance hematopoiesis during ESC differentiation, presumably by acting as developmental switches to activate posterior Hox genes. Insights into the role of the Cdx-Hox gene pathway during embryonic hematopoietic development in the zebrafish have allowed us to improve the derivation of repopulating HSCs from murine ESCs.
Towards Hematopoietic Reconstitution from Embryonic Stem Cells: a Sanguine Future
To review recent progress towards the derivation of hematopoietic stem cells (HSCs) and blood lineages from embryonic stem cells (ESCs), and to highlight the hurdles that must be overcome in order to move the field closer to a clinical application.
Modulation of Murine Embryonic Stem Cell-derived CD41+c-kit+ Hematopoietic Progenitors by Ectopic Expression of Cdx Genes
Cdx1, Cdx2, and Cdx4 comprise the caudal-like Cdx gene family in mammals, whose homologues regulate hematopoietic development in zebrafish. Previously, we reported that overexpression of Cdx4 enhances hematopoietic potential from murine embryonic stem cells (ESCs). Here we compare the effect of ectopic Cdx1, Cdx2, and Cdx4 on the differentiation of murine ESC-derived hematopoietic progenitors. The 3 Cdx genes differentially influence the formation and differentiation of hematopoietic progenitors within a CD41(+)c-kit(+) population of embryoid body (EB)-derived cells. Cdx1 and Cdx4 enhance, whereas Cdx2 strongly inhibits, the hematopoietic potential of CD41(+)ckit(+) EB-derived cells, changes that are reflected by effects on hematopoietic lineage-specific and Hox gene expression. When we subject stromal cell and colony assay cultures of EB-derived hematopoietic progenitors to ectopic expression of Cdx genes, Cdx4 dramatically enhances, whereas Cdx1 and Cdx2 both inhibit hematopoietic activity, probably by blocking progenitor differentiation. These data demonstrate distinct effects of Cdx genes on hematopoietic progenitor formation and differentiation, insights that we are using to facilitate efforts at in vitro culture of hematopoietic progenitors from ESC. The behavior of Cdx genes in vitro suggests how derangement of these developmental regulators might contribute to leukemogenesis.
BMP and Wnt Specify Hematopoietic Fate by Activation of the Cdx-Hox Pathway
The formation of blood in the embryo is dependent on bone morphogenetic protein (BMP), but how BMP signaling intersects with other regulators of hematopoietic development is unclear. Using embryonic stem (ES) cells, we show that BMP4 first induces ventral-posterior (V-P) mesoderm and subsequently directs mesodermal cells toward blood fate by activating Wnt3a and upregulating Cdx and Hox genes. When BMP signaling is blocked during this latter phase, enforced expression of either Cdx1 or Cdx4 rescues hematopoietic development, thereby placing BMP4 signaling upstream of the Cdx-Hox pathway. Wnt signaling cooperates in BMP-induced hemogenesis, and the Wnt effector LEF1 mediates BMP4 activation of Cdx genes. Our data suggest that BMP signaling plays two distinct and sequential roles during blood formation, initially as an inducer of mesoderm, and later to specify blood via activation of Wnt signaling and the Cdx-Hox pathway.
Cdx Gene Deficiency Compromises Embryonic Hematopoiesis in the Mouse
Cdx genes (Cdx1, Cdx2, and Cdx4) encode a family of caudal-related transcription factors that mediate anterior-posterior patterning during embryogenesis through Hox gene regulation. Homologues in the zebrafish have been shown to play key roles in blood formation. To define the role of Cdx genes during embryonic hematopoiesis in a mammalian system, we examined the hematopoietic potential of Cdx-deficient mouse embryonic stem cells (ESCs) in vitro and in vivo. Individual Cdx-deficient ESCs exhibited impaired embryonic hematopoietic progenitor formation and altered Hox gene expression, most notably for Cdx2 deficiency. A more severe hematopoietic defect was observed with compound Cdx deficiency than loss of function of any single Cdx gene. Reduced hematopoietic progenitor formation of ESCs deficient in multiple Cdx genes could be rescued by ectopic expression of Cdx4, concomitant with partially restored Hox gene expression. These results reveal an essential and partially redundant role for multiple Cdx genes during embryonic hematopoiesis in the mouse.
Isolation of Hematopoietic Stem Cells from Mouse Embryonic Stem Cells
This unit describes a protocol for the isolation of cells from murine embryonic stem cells with hematopoietic stem cell activity, defined by the ability to reconstitute, long term, multiple lineages of the hematopoietic system of lethally irradiated mice. The protocol subjects hematopoietic progenitors specified in differentiating embryoid bodies to ectopic HoxB4 expression (delivered via retroviral infection), followed by coculture and expansion on OP9 stromal cells in the presence of hematopoietic cytokines for 10 days. The protocol results in the generation of hundreds of millions of cells that can rescue mice from lethal irradiation. Although little is known about the phenotype and frequency of the actual hematopoietic stem cell-like cell within the population of cells generated by this protocol, the protocol establishes a system in which these cells can be further studied and the results ultimately translated to the human system.
Surface Antigen Phenotypes of Hematopoietic Stem Cells from Embryos and Murine Embryonic Stem Cells
Surface antigens on hematopoietic stem cells (HSCs) enable prospective isolation and characterization. Here, we compare the cell-surface phenotype of hematopoietic repopulating cells from murine yolk sac, aorta-gonad-mesonephros, placenta, fetal liver, and bone marrow with that of HSCs derived from the in vitro differentiation of murine embryonic stem cells (ESC-HSCs). Whereas c-Kit marks all HSC populations, CD41, CD45, CD34, and CD150 were developmentally regulated: the earliest embryonic HSCs express CD41 and CD34 and lack CD45 and CD150, whereas more mature HSCs lack CD41 and CD34 and express CD45 and CD150. ESC-HSCs express CD41 and CD150, lack CD34, and are heterogeneous for CD45. Finally, although CD48 was absent from all in vivo HSCs examined, ESC-HSCs were heterogeneous for the expression of this molecule. This unique phenotype signifies a developmentally immature population of cells with features of both primitive and mature HSC. The prospective fractionation of ESC-HSCs will facilitate studies of HSC maturation essential for normal functional engraftment in irradiated adults.
Biomechanical Forces Promote Embryonic Haematopoiesis
Biomechanical forces are emerging as critical regulators of embryogenesis, particularly in the developing cardiovascular system. After initiation of the heartbeat in vertebrates, cells lining the ventral aspect of the dorsal aorta, the placental vessels, and the umbilical and vitelline arteries initiate expression of the transcription factor Runx1 (refs 3-5), a master regulator of haematopoiesis, and give rise to haematopoietic cells. It remains unknown whether the biomechanical forces imposed on the vascular wall at this developmental stage act as a determinant of haematopoietic potential. Here, using mouse embryonic stem cells differentiated in vitro, we show that fluid shear stress increases the expression of Runx1 in CD41(+)c-Kit(+) haematopoietic progenitor cells, concomitantly augmenting their haematopoietic colony-forming potential. Moreover, we find that shear stress increases haematopoietic colony-forming potential and expression of haematopoietic markers in the para-aortic splanchnopleura/aorta-gonads-mesonephros of mouse embryos and that abrogation of nitric oxide, a mediator of shear-stress-induced signalling, compromises haematopoietic potential in vitro and in vivo. Collectively, these data reveal a critical role for biomechanical forces in haematopoietic development.
Cdx4 is Dispensable for Murine Adult Hematopoietic Stem Cells but Promotes MLL-AF9-mediated Leukemogenesis
Cdx4 is a homeobox gene essential for normal blood formation during embryonic development in the zebrafish, through activation of posterior Hox genes. However, its role in adult mammalian hematopoiesis has not been extensively studied and its requirement in leukemia associated with Hox gene expression alteration is unclear.
