Mouse hematopoietic stem cell identification and analysis (original) (raw)
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Purification and analysis of rat hematopoietic stem cells by flow cytometry
Cytometry, 1987
The monoclonal antibody OX7 recognizes an epitope expressed on the Thy-1 glycoprotein, OX22 recognizes the high molecular weight form(s) on leukocyte common antigen, and W3/13 recognized determinants found on certain sialoglycoproteins. Recently, the rat colony-forming unit spleen (CFU-S) was characterized as being OX7 upper 20% positive (OX7u20%), OX22 negative (OX22-), and W3/13 weakly positive (W3/13+). In the present study these observations have been extended to include the hematopoietic stem cell (HSC). Rat marrow cells were incubated with allophycocyanine-OX7 Fab' (APC-OX7 Fab') and phycoerythrin B-OX22 Fab' (Phy B-OX22 Fab'). The cells were sorted with a FACS-II instrument by using a Krypton laser tuned to the 530 nm spectral line for phycobiliprotein excitation. It was found that marrow cells capable of protecting lethally irradiated Lewis rats (9.5 Gy total body radiation, 0.4 Gy/min Co60) had the phenotype OXu20%, OX22-. The percentage of cells in the marr...
Hematopoietic stem cells: Inferences from in vivo assays
Stem Cells, 1997
Mice and humans both contain a population in their marrow which can permanently regenerate all of the hematopoietic lineages. This developmental potential was first demonstrated in myeloablated mice transplanted with genetically marked marrow obtained from congenic donors. More recently, this approach has been used to devise an in vivo limiting dilution assay for "competitive (lymphomyeloid) repopulating units" (CRU) that allows murine hematopoietic stem cells to be quantitated. Measurements of murine CRU have shown that this population expands concomitantly with the total hematopoietic system during ontogeny and to some extent post-transplant. During these periods of expansion, defective c-kit function can be seen to preferentially compromise CRU self-renewal more than early CRU detection (which requires differentiation and amplification of the progeny of CRU, but may not require extensive CRU selfrenewal). In humans, a similar cell type with transplantable lymphomyeloid differentiation potential can be identified in cord blood on the basis of its ability to engraft sublethally irradiated immunodeficient nonobese diabetidsevere combined immunodeficient mice. Quantitation of these human CRU by limiting dilution analysis of unseparated, highly purified (CD34TD38) and cultured (CD34'CD38-) human cord blood cells indicates that their numbers (like the longterm culture-initiating cell [LTC-IC] population) can be slightly expanded in cytokine-supplemented serum-free media, although not as extensively as anticipated from analogous studies of human marrow LTC-IC cultured under the same conditions. Taken together, the results of our studies suggest that the self-renewal of mitotically activated hematopoietic stem cells can be enhanced by their interactions with particular cytokine combinations whose effectiveness in this regard may change during ontogeny. Stem Cells 1997;15(supp1 1):1-5 Dr. Dick: Do you think a cell that reads out at 12 weeks is going to be a different cell when it reads out in 6 weeks? Dr. C. Eaves: I guess the question is, "Does it matter in terms of the programming of the cell, in terms of readouts that we're interested in, and is it a reversible phenomenon?' We do not h o w .
Methods in Enzymology, 2006
Hematopoietic stem cells (HSCs) have the capacity to selfârenew and the potential to differentiate into all of the mature blood cell types. The ability to prospectively identify and isolate HSCs has been the subject of extensive investigation since the first transplantation studies implying their existence almost 50 years ago. Despite significant advances in enrichment protocols, the continuous in vitro propagation of human HSCs has not yet been achieved. This chapter describes current procedures used to phenotypically and functionally characterize candidate human HSCs and initial efforts to derive permanent human HSC lines.
Analysis of hematopoietic stem cells using a composite approach
The International Journal of Biochemistry & Cell Biology, 2019
Stem cells or Cancer stem cells (CSCs) have now been identified in different type of tissues by using surface markers. Functional assays such as ALDEFLUOR and side population which are marker independent have been additional approaches. However, whether all these approaches identify the same population of cells remain uncertain. To address this issue we have used hematopoietic stem cells as a model. Peripheral blood stem cells enumerated by CD34 are used routinely in bone marrow transplantation which supports the recovery of bone marrow after ablative chemotherapy or radiation. Hematopoietic stem cells (HSCs) were obtained from normal donor bone marrow (n=5) and G-CSF stimulated peripheral blood stem cells (PBSCs) (n=5) from patients undergoing leukapheresis prior to bone marrow transplantation. The stem cells were identified by combining CD34 expression with functional assays (ALDEFLUOR and side population). The cell cycle profile was further determined by simultaneous labeling of these cells with Hoechst and Pyronin Y. The simultaneous analysis showed that both CD34+ and CD34-cells co-exist with ALDH1A1+ cells but side population did not segregate with CD34+ cells. Though stem cell populations identified by functional assays were different, the cell cycle analysis showed that both ALDH1A1+ and CD34+ cells were in the G1 phase of cell cycle rather than in the quiescent (G0) phase.
Molecular and cellular basis of hematopoietic stem cells maintenance and differentiation
The blood system consists of two main lineages: myeloid and lymphoid. The myeloid system consists of cells that are part of the innate immune response while the lymphoid system consist of cells that are part of humoral response. These responses protect our bodies from foreign pathogens. Thus, malignancies in these systems often cause complications and mortality. Scientists worldwide have been researching alternatives to treat hematologic disorders and have explored induced pluripotent stem cells (iPSCs) and the conversion of one cell type to another. First, iPSCs were generated by overexpression of four transcription factors: Oct4, Sox2, Klf4 and cMyc. These cells closely resemble embryonic stem cells (ESCs) at the molecular and cellular level. However, the efficiency of cell conversion is less than 0.1%. In addition, many iPS colonies can arise from the same culture, but each has a different molecular signature and potential. Identifying the appropriate iPSC lines to use for patient specific therapy is crucial. Here we demonstrate that our system is highly efficient in generating iPSC lines, and cell lines with silent transgenes are most efficient in differentiating to different cell types. Second, we are interested in generating hematopoietic stem cells (HSCs) from fibroblasts directly, without going through the pluripotent state, to increase efficiency and to avoid complications associated with a stem cell intermediate. However, a robust hematopoietic reporter system remains elusive. There are multiple hematopoietic reporter candidates, but we demonstrate that the CD45 gene was the most promising. CD45 is expressed early during hematopoiesis on the surface of HSCs; and as HSCs differentiate CD45 levels increase. Furthermore, the CD45 reporter is only active in hematopoietic vii cells. We were able to confirm the utility of the CD45 reporter using an in vitro and an in vivo murine model. In conclusion, the goal of this research was to expand the knowledge of stem cell reprogramming, specifically the reprogramming of iPSCs. Furthermore, it is our desire that the CD45 reporter system will undergo further validation and find utility in clinical and cell therapy environments. viii
The hematopoietic stem compartment consists of a limited number of discrete stem cell subsets
Blood, 2006
Hematopoietic stem cells (HSC) display extensive heterogeneity in their behavior even when isolated as phenotypically homogeneous populations. It is not clear, whether this heterogeneity reflects inherently diverse subsets of HSC or a homogeneous population of HSC diversified by their response to different external stimuli. To address this, we analyzed 97 individual HSC in long-term transplantation assays. HSC clones were obtained from unseparated bone marrow (BM) through limiting dilution approaches. Following transplantation into individual hosts, donor type cells in blood were measured bimonthly and the resulting repopulation kinetics were grouped according to overall shape. Only 16 types of repopulation kinetics were found amongst the HSC clones even though combinatorically 54 groups were possible. All HSC clones, regardless of their origin, could be assigned to this subset of groups and the probability of finding new patterns is negligible. Thus, the full repertoire of repopulating HSC was covered. These data indicate that the HSC compartment consists of a limited number of distinct HSC subsets, each with predictable behavior. Enrichment of HSC (Lin-Rho-SP) changes the representation of HSC types by selecting for distinct subsets of HSC. These data from the steady-state HSC repertoire could provide a basis for the diagnosis of perturbed patterns of HSC potentially caused by disease or aging.