CD27, CD201, FLT3, CD48, and CD150 cell surface staining identifies long-term mouse hematopoietic stem cells in immunodeficient non-obese diabetic severe combined immune deficient-derived strains

Abstract The cell surface protein CD34 is frequently used as a marker for positive selection of human hematopoietic stem/progenitor cells in research and in transplantation. However, populations of reconstituting human and murine stem cells that lack cell surface CD34 protein have been identified. In the current studies, we demonstrate that CD34 expression is reversible on human hematopoietic stem/progenitor cells. We identified and functionally characterized a population of human CD45+/CD34− cells that was recovered from the bone marrow of immunodeficient beige/nude/xid (bnx) mice 8 to 12 months after transplantation of highly purified human bone marrow–derived CD34+/CD38− stem/progenitor cells. The human CD45+ cells were devoid of CD34 protein and mRNA when isolated from the mice. However, significantly higher numbers of human colony-forming units and long-term culture-initiating cells per engrafted human CD45+ cell were recovered from the marrow of bnx mice than from the marrow of human stem cell–engrafted nonobese diabetic/severe combined immunodeficient mice, where 24% of the human graft maintained CD34 expression. In addition to their capacity for extensive in vitro generative capacity, the human CD45+/CD34− cells recovered from thebnx bone marrow were determined to have secondary reconstitution capacity and to produce CD34+ progeny following retransplantation. These studies demonstrate that the human CD34+ population can act as a reservoir for generation of CD34− cells. In the current studies we demonstrate that human CD34+/CD38− cells can generate CD45+/CD34− progeny in a long-term xenograft model and that those CD45+/CD34− cells can regenerate CD34+ progeny following secondary transplantation. Therefore, expression of CD34 can be reversible on reconstituting human hematopoietic stem cells.

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CD150− side population cells represent a functionally distinct population of long-term hematopoietic stem cells

Blood ◽

10.1182/blood-2007-09-115006 ◽

2008 ◽

Vol 111 (4) ◽

pp. 2444-2451 ◽

Cited By ~ 94

Author(s):

David C. Weksberg ◽

Stuart M. Chambers ◽

Nathan C. Boles ◽

Margaret A. Goodell

Keyword(s):

Stem Cells ◽

Cell Surface ◽

Hematopoietic Stem Cells ◽

Cell Biology ◽

Side Population ◽

Surface Markers ◽

Cell Surface Markers ◽

Hematopoietic Stem ◽

Slam Family

Hematopoietic stem cells (HSCs) are a self-renewing population of bone marrow cells that replenish the cellular elements of blood throughout life. HSCs represent a paradigm for the study of stem-cell biology, because robust methods for prospective isolation of HSCs have facilitated rigorous characterization of these cells. Recently, a new isolation method was reported, using the SLAM family of cell-surface markers, including CD150 (SlamF1), to offer potential advantages over established protocols. We examined the overlap between SLAM family member expression with an established isolation scheme based on Hoechst dye efflux (side population; SP) in conjunction with canonical HSC cell-surface markers (Sca-1, c-Kit, and lineage markers). Importantly, we find that stringent gating of SLAM markers is essential to achieving purity in HSC isolation and that the inclusion of canonical HSC markers in the SLAM scheme can greatly augment HSC purity. Furthermore, we observe that both CD150+ and CD150− cells can be found within the SP population and that both populations can contribute to long-term multilineage reconstitution. Thus, using SLAM family markers to isolate HSCs excludes a substantial fraction of the marrow HSC compartment. Interestingly, these 2 subpopulations are functionally distinct, with respect to lineage output as well as proliferative status.

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Plxdc2 Marks Hematopoietic Stem Cells and Th2 Cytokine-Producing Innate Lymphocytes in Adult Bone Marrow

Blood ◽

10.1182/blood.v118.21.1278.1278 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1278-1278

Author(s):

Yasushi Kubota ◽

Ivo Lieberam ◽

Shinya Kimura ◽

Thomas M Jessell ◽

Shin-Ichi Nishikawa

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

T Cell ◽

Cell Surface ◽

Hematopoietic Stem Cells ◽

Cell Population ◽

Specific Marker ◽

Simple Method ◽

Hematopoietic Stem

Abstract Abstract 1278 Hematopoietic stem cells (HSCs) have been highly enriched using combinations of more than 10 surface markers. However the simple method using a few positive markers is preferable to identify HSCs location in tissue section. We performed a stringent comparative gene expression profiling analysis to find genes preferentially expressed in the HSC population, and identified a total of 63 genes that are highly expressed in HSC among various hematopoietic cell population. In order to find HSC-specific marker we focused on genes encoding cell surface protein, and found that plexin domain containing 2 (Plxdc2) is highly expressed in CD34—c-Kit+Sca-1+Lineage−(CD34−KSL) HSC population using Plxdc2::GFP knock-in mice. Only 0.2% of whole bone marrow cells were Plxdc2+, and competitive repopulation assay clearly showed that all HSCs are included in the Plxdc2+ fraction. These results identify Plxdc2 as a new marker of HSCs. Plxdc2+ population contain not only HSCs but uncharacterized c-Kitlow/−Sca-1+Lineage−cells. To further purify HSCs, we investigated the additional positive marker. Throughout the screening of various known HSC-related marker, CD150 was selected. CD150 is already recognized as a positive HSC marker (Kiel, et al. Cell 2005). The Plxdc2+CD150+ fraction represented only 0.1%±0.002% in whole bone marrow, and 6% in c-Kit+Sca-1+Lineage− cells, respectively. To test whether the combination of Plxdc2 and CD150 with or without other markers can highly enrich long-term HSCs, we competitively reconstituted irradiated mice with single Plxdc2+CD150+ cells or single Plxdc2+CD150+c-Kit+Sca-1+Lineage− cells. One out of every 4.6 Plxdc2+CD150+ cells (22%), and one out of 2.2 Plxdc2+CD150+c-Kit+Sca-1+Lineage− cells (44%) engrafted and gave long-term multi-lineage reconstitution. The simple combination of Plxdc2 and CD150 significantly increased HSC purity. In addition, we found robust levels of PLXDC2 transcripts in purified human cord blood CD34+ HSCs. Next, we attempted to characterize the another Plxdc2+ fraction which is c-Kitlow/−Sca-1+Lineage−. Multicolor flowcytometric analysis revealed that Plxdc2+c-Kitlow/−Sca-1+Lineage− cells uniformly express CD45, IL7Rα, Thy-1.2, CD27, T1/ST2 (IL1RL1, a subunit of IL33R) and CD25. These cell surface phenotype indicated that this population is probably of lymphoid lineage. However, culturing Plxdc2+ c-Kit low/−Sca-1+Lineage− cells on OP9-DL1, which supports the development of T-cell progenitors to mature T-cells, did not induce T-cell differentiation. Plxdc2+c-Kitlow/−Sca-1+Lineage−cells also did not differentiate into B cells when co-cultured with OP9 stroma cell line. Furthermore Plxdc2+c-Kitlow/−Sca-1+Lineage− cells produce IL-5 and IL-13 in response to IL-33 or a combination of IL-2 and IL-25. These characteristics resemble that of “natural helper (NH) cells”, a recently identified cell population capable of producing large amounts of Th2 cytokines in fat-associated lymphoid clusters (Moro, et al. Nature 2010). Immunohistochemical staining of bone section to detect HSCs, and functional analyses to clarify why Plxdc2 specifically express in HSCs and bone marrow “NH cells” using Plxdc2-deficient mice are our ongoing tasks. Disclosures: No relevant conflicts of interest to declare.

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Robo4/Magic Roundabout Is a Novel Surface Marker for Murine and Human Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v108.11.682.682 ◽

2006 ◽

Vol 108 (11) ◽

pp. 682-682

Author(s):

Fumi Shibata ◽

Yuko Goto-Koshino ◽

Miyuki Ito ◽

Yumi Fukuchi ◽

Yoshihiro Morikawa ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cell Surface ◽

Hematopoietic Stem Cells ◽

Surface Markers ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Cd34 Cells ◽

Human Hscs

Abstract A variety of cell surface markers such as c-Kit, Sca-1, CD34 and Flt-3 have been utilized to prospectively isolate murine or human hematopoietic stem cells (HSCs). While murine HSCs were shown to be highly enriched in CD34−c-Kit+Sca-1+Lineage- (CD34−KSL) fraction, this population is still not homogeneous for long-term HSCs. In human, CD34+ cells are regarded as crude HSC fraction and used for clinical applications. However, quiescent human HSCs are also found in CD34− fraction, indicating that CD34 is not a bona fide marker for human HSC. Thus, novel surface markers that can be used to purify human or murine HSCs to homogeneity need to be identified. Roundabout (Robo) family proteins are immunoglobulin-type cell surface receptors that are predominantly expressed in nervous system. Slit2, a ligand for Robo, is a large leucine-rich repeat-containing secreted protein that is also expressed in brain. By binding with Robo, Slit2 acts as a repellant for axon guidance of developing neurons and they are critical for correct wiring of neuronal network. Robo family comprises four family members, Robo1 – Robo4, and Robo4 is distinct in that it is expressed specifically in endothelial cells, but not in brain. In this study, we investigated Robo4 for its possible application for HSC identification in murine and human hematopoietic system. By RT-PCR, Robo4 was specifically expressed in murine KSL fraction, and was not expressed in lineage positive cells and various progenitors such as common myeloid progenitor (CMP), granulocyte-monocyte progenitor (GMP), megakaryocyte/erythroid progenitor (MEP) and common lymphoid progenitor (CLP). Moreover, the expression of Robo4 was highest in side population of KSL cells (KSL-SP), and moderate in KSL-main population (KSL-MP) cells. Monoclonal antibody raised against Robo4 identified its high expression in KSL cells by FACS. FACS analysis of human cord blood cells revealed that Robo4 is highly expressed in CD34+ cells, and CD34+Robo4high population fell into CD38− fraction, which enriches human HSCs. Bone marrow transplantation experiments revealed that Robo4+ fraction of murine KSL cells had long-term repopulating activity, while Robo4−KSL cells not. Although both Robo4+ and Robo4− CD34−KSL cells repopulated murine hematopoietic system for long-term, Robo4+CD34−KSL cells achieved higher chimerism after repopulation compared with Robo4−CD34−KSL. To investigate the physiological role of Robo4 in HSC homeostasis, we next examined the expression of Slit2 in hematopoietic system. Interestingly, Slit2 is specifically expressed in bone marrow stromal cells, but not in hematopoietic cells. Moreover, Slit2 is induced in osteoblasts, a critical cellular component composing HSC niche, in response to myelosuppressive stress such as 5FU treatment. These results indicate that Robo4 is expressed in murine and human hematopoietic HSCs and useful for HSC purification, and Robo4 - Slit2 system may play a role in HSC physiology in niche environment under hematopoietic stress.

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Long-term repopulation of irradiated mice with limiting numbers of purified hematopoietic stem cells: in vivo expansion of stem cell phenotype but not function

Blood ◽

10.1182/blood.v85.4.1006.bloodjournal8541006 ◽

1995 ◽

Vol 85 (4) ◽

pp. 1006-1016 ◽

Cited By ~ 245

Author(s):

GJ Spangrude ◽

DM Brooks ◽

DB Tumas

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Cell Surface ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Antigen Expression ◽

Hematopoietic Stem ◽

Surface Antigen Expression

Hematopoietic stem cells were isolated from normal adult mouse bone marrow based on surface antigen expression (Thy-1.1(low)Lin(neg)Ly- 6A/E+) and further selected for low retention of rhodamine 123. This population of cells (Rh-123low) could mediate radioprotection and long- term (greater than 12 months) repopulation after transplantation of as few as 25 cells. Transfer of five genetically marked Rh-123low cells in the presence of 10(5) normal bone marrow cells resulted in reconstitution of peripheral blood by greater than 10% donor cells in 64% (30 of 47) of recipient mice. Of 46 animals surviving after 24 weeks, 10 had over 50% donor-derived cells in peripheral blood. Two general patterns of long-term reconstitution were observed: one in which many donor-derived cells were observed 5 to 6 weeks after reconstitution and another in which donor-derived cells were rare initially but expanded with time. This result suggests that two classes of long-term repopulating hematopoietic stem cells exist, differing in their ability to function early in the course of transplantation. Alternatively, distinct anatomic sites of engraftment may dictate these two outcomes from a single type of cell. As an approach to measure the extent of self-renewal by the injected cells, recipients of five or 200 stem cells were killed 8 to 13 months after the transplants, and Thy- 1.1(low)Lin(neg)Ly-6A/E+ progeny of the original injected cells were isolated for a second transplant. While a numerical expansion of cells expressing the cell surface phenotype of stem cells was observed, along with activity in the colony-forming unit-spleen assay, the expanded cells were vastly inferior in radioprotection and long-term reconstitution assays when compared with cells freshly isolated from normal animals. This result demonstrates that in stem cell expansion experiments, cell surface antigen expression is not an appropriate indicator of stem cell function.

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Faculty Opinions recommendation of Cultivation of aorta-gonad-mesonephros-derived hematopoietic stem cells in the fetal liver microenvironment amplifies long-term repopulating activity and enhances engraftment to the bone marrow.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1004105.47154 ◽

2002 ◽

Author(s):

Ana Cumano

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Fetal Liver ◽

Hematopoietic Stem

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Faculty Opinions recommendation of Isolation of single human hematopoietic stem cells capable of long-term multilineage engraftment.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.12101960.14635181 ◽

2011 ◽

Author(s):

Timm Schroeder ◽

Michael Rieger

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem

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Faculty Opinions recommendation of Isolation of single human hematopoietic stem cells capable of long-term multilineage engraftment.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.12101960.13421055 ◽

2011 ◽

Author(s):

Dan Kaufman ◽

David Knorr

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem

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Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

Current Stem Cell Research & Therapy ◽

10.2174/1574888x15666200225091339 ◽

2020 ◽

Vol 15 ◽

Author(s):

Fatima Aerts-Kaya

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Ex Vivo ◽

Stress Conditions ◽

Stress Factors ◽

Hematopoietic Stem ◽

Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

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