Isolation and Assessment of Long‐Term Reconstituting Hematopoietic Stem Cells from Adult Mouse Bone Marrow

Kent, David G.; Dykstra, Brad; Eaves, Connie J.

doi:10.1002/9780470151808.sc02a04s3

Cited by 20 publications

(13 citation statements)

References 59 publications

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“…For all transplantation assays, recipients were C57BL/6J (CD45.1) mice irradiated with a split dose (2×475 cGy) and all transplants were performed by standard intravenous tail vein injection using a 29.5G insulin syringe. Peripheral blood was collected and analyzed as described previously [64].…”

Section: Methodsmentioning

confidence: 99%

“…For all transplantation assays, peripheral blood samples were collected from the tail vein of mice at 4, 8, and 16 wk after transplantation and analyzed for repopulation levels as described previously [64]. Antibodies used were CD45.1-PE (Clone A20, Biolegend), CD45.2-FITC (Clone 104, BD), Ly6g-Pacific Blue (Clone RB6-8C5, Biolegend), Mac1-APC (Clone M1/70, Biolegend), B220-APC (Clone RA3-6B2 eBiosciences, San Diego, CA), and CD3e-Pacific Blue (Clone 500A2, BD).…”

Section: Methodsmentioning

confidence: 99%

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Self-Renewal of Single Mouse Hematopoietic Stem Cells Is Reduced by JAK2V617F Without Compromising Progenitor Cell Expansion

Kent

Tanna

et al. 2013

PLoS Biol

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Self-Renewal of Single Mouse Hematopoietic Stem Cells Is Reduced by JAK2V617F Without Compromising Progenitor Cell Expansion

Kent

Tanna

et al. 2013

PLoS Biol

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“…All experiments used C57BL/6J (B6)-Ly5.1 or -Ly5.2 mice as donors and congenic sublethally irradiated B6-W 41 /W 41 -Ly5.1 and -Ly5.2 (W 41 -5.1 and W 41 -5.2) mice as recipients according to institutionally approved procedures. Transplantations and analyses of transplanted mice were performed as previously described (Kent et al, 2007). The HSC subtype origin of each clone was first determined 16 weeks posttransplant and for secondary transplants was reconfirmed within 1 week of the secondary transplantation.…”

Section: Experimental Procedures Micementioning

confidence: 99%

Hematopoietic Stem Cell Subtypes Expand Differentially during Development and Display Distinct Lymphopoietic Programs

et al. 2012

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Adult hematopoietic stem cells (HSCs) with serially transplantable activity comprise two subtypes. One shows a balanced output of mature lymphoid and myeloid cells; the other appears selectively lymphoid deficient. We now show that both of these HSC subtypes are present in the fetal liver (at a 1:10 ratio) with the rarer, lymphoid-deficient HSCs immediately gaining an increased representation in the fetal bone marrow, suggesting that the marrow niche plays a key role in regulating their ensuing preferential amplification. Clonal analysis of HSC expansion posttransplant showed that both subtypes display an extensive but variable self-renewal activity with occasional interconversion. Clonal analysis of their differentiation programs demonstrated functional and molecular as well as quantitative HSC subtype-specific differences in the lymphoid progenitors they generate but an indistinguishable production of multipotent and myeloid-restricted progenitors. These findings establish a level of heterogeneity in HSC differentiation and expansion control that may have relevance to stem cell populations in other hierarchically organized tissues.

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“…23 Peripheral blood samples were collected from the tail vein of some mice at 4 weeks and all mice at 8, 16, and 24 weeks after transplantation and RBCs lysed using NH 4 Cl. All samples were stained with both anti-CD45.1-APC (eBiosciences, San Diego, CA) and anti-CD45.2-FITC (Terry Fox Laboratory) to enable donor and recipient WBCs to be distinguished and to ensure that any double positive CD45.1 and CD45.2 events were excluded from the analysis of donor contributions to specific WBC subsets.…”

Section: Transplantation Of Cells and Analysis Of Mice That Received mentioning

confidence: 99%

Prospective isolation and molecular characterization of hematopoietic stem cells with durable self-renewal potential

Kent

Copley

Benz

et al. 2009

Blood

311

290

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IntroductionEach day, the hematopoietic system of the adult mouse produces billions of mature blood cells. The multistep process that underlies the production of these cells is controlled by complex mechanisms that enable changing physiologic demands to be met without overwhelming the system. It is now clear that a small population of cells known as hematopoietic stem cells (HSCs) are ultimately responsible for maintaining the lifelong output of new blood cells. 1 Nevertheless, a molecular understanding of what constitutes an HSC and how its key functions are maintained are still poorly understood.The existence of hematopoietic cells with the individual potential to produce large numbers of multiple blood cell types in vivo for prolonged periods was first suggested by retrospective clonal tracking experiments that used unique chromosomal, 2 and later, retroviral marking approaches. 3,4 The relatively short lifespan of many mature blood cell types and the contrasting longevity of some of the multilineage clones identified in these experiments implied an origin of the clones from undifferentiated cells with an extensive ability to divide and maintain a derivative population of similarly undifferentiated cells. Serial transplantation experiments demonstrated that such cell divisions did occur and thus provided the first definitive evidence of HSC self-renewal. 3,4 These findings prompted a search for functional end points that would allow HSCs to be specifically quantified independent of the presence of other cell types when assayed in limiting dilution transplantation strategies using suitably irradiated congenic hosts. 5,6 A sustained output of at least 1% of all the circulating white blood cells (WBCs) for at least 4 months is now widely assumed to be suitable for this purpose. 7 Interestingly, most analyses of individual pluripotent hematopoietic cells proliferating either in vivo or in vitro have generally found the self-renewal activity actually displayed to be highly variable. 3,4,8,9 How such a variable behavior is related to the molecular state of the initial cells is not well defined and remains a subject of intense interest and investigation. 10 Variations in external cues may be one contributing parameter, at least in vivo, because it is known that self-renewal responses can be directly and rapidly modulated in this way in vitro. 11,12 In addition, there is some evidence of predetermined heterogeneity in HSC self-renewal potential. This is exemplified by the differences in regenerative activity of HSCs from fetal and adult sources 13,14 and the finding of a consistent association of short-term and long-term multilineage WBC outputs with distinct phenotypes of adult bone marrow (BM) cells (eg, according to their expression of CD49b and CD34). 15,16 Taken together, these results suggest that some hematopoietic cells can remain pluripotent for several divisions even though they are already destined to undergo terminal differentiation within a finite period. This is the basis of the concept of durable versu...

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Isolation and Assessment of Long‐Term Reconstituting Hematopoietic Stem Cells from Adult Mouse Bone Marrow

Cited by 20 publications

References 59 publications

Self-Renewal of Single Mouse Hematopoietic Stem Cells Is Reduced by JAK2V617F Without Compromising Progenitor Cell Expansion

Self-Renewal of Single Mouse Hematopoietic Stem Cells Is Reduced by JAK2V617F Without Compromising Progenitor Cell Expansion

Hematopoietic Stem Cell Subtypes Expand Differentially during Development and Display Distinct Lymphopoietic Programs

Prospective isolation and molecular characterization of hematopoietic stem cells with durable self-renewal potential

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