Changing phenotypes of hematopoietic stem cells.

Abstract

Identification of the surface phenotypes of hematopoietic stem cells is an important subject in both clinical stem cell transplantation and basic research in hematopoiesis. Transplantation of pure populations of stem cells should eliminate the occurrence of graft-vs-host disease (GVHD) in allogeneic transplantation and may reduce the recurrence rate of malignancies in autologous transplantation. Availability of highly enriched populations of stem cells should facilitate investigations in a number of areas such as in vitro expansion, cryogenic storage, and gene therapy using stem cells. An ultimate goal is to develop a method to quantitate the number of living stem cells based on their surface characteristics. During the last two decades, investigators identified a number of surface molecules, which, in combinations, were thought to provide exact definition of murine and human stem cells. Recent studies of murine stem cells, however, clearly demonstrated that expression of the surface antigens of stem cells is under the influence of developmental stages and the kinetic state of the stem cells. This review summarizes the concept of the changing phenotypes of hematopoietic stem cells. For more than a decade, CD34 has been the best-known marker of hematopoietic stem cells, generating academic as well as commercial interest. In 1988 Berenson et al. [1Berenson R.J Andrews R.G Bensinger W.I et al.Antigen CD34+ marrow cells engraft lethally irradiated baboons.J Clin Invest. 1988; 81: 951Crossref PubMed Scopus (476) Google Scholar], using anti-human CD34 antibody 12.8, reported successful hematopoietic reconstitution in baboons with selected CD34+ bone marrow cells. Subsequently, a number of clinical studies testing allogeneic transplantation of selected CD34+ cells clearly documented the presence of CD34+ human stem cells [2Bensinger W.I Buckner C.D Shannon-Dorcy K et al.Transplantation of allogeneic CD34+ peripheral blood stem cells in patients with advanced hematologic malignancy.Blood. 1996; 88: 4132PubMed Google Scholar, 3Link H Arseniev L Bahre O Kadar J.G Diedrich H Poliwanda H Transplantation of allogeneic CD34+ blood cells.Blood. 1996; 87: 4903PubMed Google Scholar, 4Hassan H.T Zeller W Stockschlader M Kruger W Hoffknecht M.M Zander A.R Comparison between bone marrow and G-CSF–mobilized peripheral blood allografts undergoing clinical scale CD34+ cell selection.Stem Cells. 1996; 14: 419Crossref PubMed Scopus (33) Google Scholar, 5Kawano Y Takaue Y Watanabe A et al.Partially mismatched pediatric transplants with allogeneic CD34+ blood cells from a related donor.Blood. 1998; 92: 3123PubMed Google Scholar]. Studies with polyclonal anti-mouse CD34 antibodies demonstrated that CD34+ murine bone marrow cells also are capable of hematopoietic reconstitution in lethally irradiated mice [6Krause D.S Ito T Fackler M.J et al.Characterization of murine CD34, a marker for hematopoietic progenitor and stem cells.Blood. 1994; 84: 691Crossref PubMed Google Scholar]. These findings, together with the observation that all hematopoietic progenitors assayable in culture express CD34, firmly established the notion that hematopoietic stem cells are CD34+. Therefore, a major controversy arose when investigators in several laboratories reported that significant populations of stem cells in the bone marrow of normal adult mice do not express CD34. First, Osawa et al. [7Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell.Science. 1996; 273: 242Crossref PubMed Scopus (1699) Google Scholar], using monoclonal anti-CD34 antibody RAM34, described that only the CD34− fraction of lineage (Lin)−, Ly-6A/E (Sca-1)+, c-kit+ bone marrow cells of normal adult mice are capable of long-term hematopoietic reconstitution of lethally irradiated mice. Using the same antibody, Morel et al. [8Morel F Szilvassy S.J Travis M Chen B Galy A Primitive hematopoietic cells in murine bone marrow express the CD34 antigen.Blood. 1996; 88: 3774PubMed Google Scholar, 9Morel F Galy A Chen B Szilvassy S.J Equal distribution of competitive long-term repopulating stem cells in the CD34+ and CD34− fractions of Thy-1lowLin−/lowSca-1+ bone marrow cells.Exp Hematol. 1998; 26: 440PubMed Google Scholar] and Donnelly et al. [10Donnelly D.S Zelterman D Sharkis S Krause D.S Functional activity of murine CD34+ and CD34− hematopoietic stem cell populations.Exp Hematol. 1999; 27: 788Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar] reported that stem cells are present in both CD34+ and CD34− populations of the bone marrow cells from normal mice. Independently from these studies, reports from Goodell et al. [11Goodell M.A Brose K Paradis G Conner A.S Mulligan R.C Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo.J Exp Med. 1996; 183: 1797Crossref PubMed Scopus (2454) Google Scholar, 12Goodell M.A Rosenzweig M Kim H et al.Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species.Nat Med. 1997; 3: 1337Crossref PubMed Scopus (944) Google Scholar] also raised serious questions about CD34 expression by stem cells of mice and other animals. They observed that side population (SP) cells, which are defined by two-color flow cytometry with Hoechst 33342 dye and are highly enriched for hematopoietic stem cells, are CD34−/low. Finally, studies using two xenotransplantation methods indicated that CD34− human marrow cell populations contain long-term engrafting cells [13Zanjani E.D Almeida-Porada G Livingston A.G Flake A.W Ogawa M Human bone marrow CD34− cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells.Exp Hematol. 1998; 26: 353PubMed Google Scholar, 14Bhatia M Bonnet D Murdoch B Gan O.I Dick J.E A newly discovered class of human hematopoietic cells with SCID-repopulating activity.Nat Med. 1998; 4: 1038Crossref PubMed Scopus (523) Google Scholar]. These observations raised a possibility that we may inadvertently discard significant numbers of stem cells by CD34 selection of human samples. In our laboratory we carried out a series of transplantation studies in mice in order to clarify the controversy. In the majority of experiments, we used cells from CD45.1 C57BL/6 mice as test cells and CD45.2 mice as recipients. Following lethal total-body irradiation of the recipient mice, we transplanted CD45.1 test cells together with “compromised” marrow cells from CD45.2 mice. The “compromised” marrow cells that are prepared by two rounds of transplantation are enriched for progenitors and prevent postradiation death of the recipient mice [15Szilvassy S.J Humphries R.K Lansdorp P.M Eaves A.C Eaves C.J Quantitative assay for totipotent reconstituting hematopoietic stem cells by a competitive repopulation strategy.Proc Natl Acad Sci U S A. 1990; 87: 8736Crossref PubMed Scopus (348) Google Scholar]. The first hypothesis we tested was that stem cell CD34 expression is affected by the kinetic state of the stem cells [16Sato T Laver J.H Ogawa M Reversible expression of CD34 by murine hematopoietic stem cells.Blood. 1999; 94: 2548PubMed Google Scholar]. First, we confirmed the report from Osawa et al. [7Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell.Science. 1996; 273: 242Crossref PubMed Scopus (1699) Google Scholar] that the long-term engrafting cells in the Lin−c-kit+Sca-1+ marrow cells of normal adult mice are CD34−. We then discovered that, following injection of 150 mg/kg 5-fluorouracil (5-FU) to mice, stem cells may be found in both CD34− and CD34+ populations of the bone marrow. These results indicated that activated stem cells express CD34. This concept was corroborated by cell culture experiments with Lin−c-kit+Sca-1+ CD34− bone marrow cells in which CD34− stem cells became CD34+ after incubation for 1 week with interleukin-11 (IL-11) and steel factor (SF). Finally, we tested reversal of CD34+ stem cells to CD34− stem cells. We transplanted CD45.1 CD34+ post–5-FU marrow cells into CD45.2 primary recipients and, 3 months later, tested the CD45.1 cells in the bone marrow of the primary recipients for their engrafting capabilities in secondary CD45.2 recipients. The majority of the CD45.1 stem cells were in the CD34− cell fraction, indicating reversal of the phenotype of stem cells from CD34+ to CD34−. These observations clearly demonstrated that CD34 expression reflects the activation/kinetic state of hematopoietic stem cells and that it is reversible [16Sato T Laver J.H Ogawa M Reversible expression of CD34 by murine hematopoietic stem cells.Blood. 1999; 94: 2548PubMed Google Scholar]. It has been generally believed that mobilized blood stem cells express CD34. However, it has also been reported that mobilized stem cells and progenitors of mice are in G0/G1 phases of the cell cycle [17Roberts A.W Metcalf D Noncycling state of peripheral blood progenitor cells mobilized by granulocyte colony-stimulating factor and other cytokines.Blood. 1995; 86: 1600PubMed Google Scholar, 18Uchida N He D Friera A.M et al.The unexpected G0/G1 cell-cycle status of mobilized hematopoietic stem cells from peripheral blood.Blood. 1997; 89: 465Crossref PubMed Google Scholar]. Therefore, it was possible that dormant stem cells in the blood may not express CD34. In the next set of experiments we studied CD34 expression by granulocyte colony-stimulating factor (G-CSF)–mobilized stem cells again using the mouse transplantation model [19Tajima F Sato T Laver J.H Ogawa M CD34 expression by murine hematopoietic stem cells mobilized by G-CSF.Blood. 2000; 96: 1989PubMed Google Scholar]. We injected CD45.1 mice with 125 μg/kg of G-CSF at 12-hour intervals for 5 consecutive days and, three hours after the last G-CSF injection, harvested peripheral blood. We prepared CD34− and CD34+ populations of the nucleated blood cells by sorting and assayed each population for long-term engrafting cells in CD45.2 recipients. The majority of the stem cells were in the CD34+ population. The CD34 expression by mobilized stem cells also was reversible because retransplantation, at 8 months after the primary transplantation, of CD45.1 CD34− marrow cells from the primary recipients of CD34+ mobilized stem cells revealed long-term engraftment. We concluded from these observations that mobilized stem cells express CD34, return to the bone marrow, and become CD34− when the bone marrow achieves steady state [19Tajima F Sato T Laver J.H Ogawa M CD34 expression by murine hematopoietic stem cells mobilized by G-CSF.Blood. 2000; 96: 1989PubMed Google Scholar]. It has been documented earlier that fetal murine hematopoietic stem cells are CD34+ [20Sanchez M.J Holmes A Miles C Dzierzak E Characterization of the first definitive hematopoietic cells in the AGM and fetal livers of the mouse embryo.Immunity. 1996; 5: 513Abstract Full Text PDF PubMed Scopus (248) Google Scholar, 21Yoder M.C Hiatt K Dutt P Mukherjee P Bodine D.M Orlic D Characterization of definitive lymphohematopoietic stem cells in the day-9 murine yolk sac.Immunity. 1997; 7: 335Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar]. As presented above, studies [7Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell.Science. 1996; 273: 242Crossref PubMed Scopus (1699) Google Scholar, 16Sato T Laver J.H Ogawa M Reversible expression of CD34 by murine hematopoietic stem cells.Blood. 1999; 94: 2548PubMed Google Scholar] of normal adult mice clearly demonstrated that adult stem cells in the steady-state marrow are CD34−. In the next series of experiments we examined the time of ontogenetic changes in the stem cell CD34 expression [22Ito T Tajima F Ogawa M Developmental changes of CD34 expression by murine hematopoietic stem cells.Exp Hematol. 2000; 28: 1269Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar]. We carried out systematic analyses of long-term engrafting cells in the bone marrow and/or blood of neonatal, juvenile, and adult mice. In order to obtain information on the total number of stem cells, we divided mononuclear cells directly into CD34− and CD34+ populations and assayed their long-term reconstituting abilities by transplantation into lethally irradiated CD45-congenic mice. All stem cells from neonatal to 5-week-old mice were CD34+ [22Ito T Tajima F Ogawa M Developmental changes of CD34 expression by murine hematopoietic stem cells.Exp Hematol. 2000; 28: 1269Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar]. In 7-week-old mice, CD34− stem cells began to emerge, and the majority of the stem cells were CD34− in the 10- and 20-week-old mice. Calculation of competitive repopulating units [15Szilvassy S.J Humphries R.K Lansdorp P.M Eaves A.C Eaves C.J Quantitative assay for totipotent reconstituting hematopoietic stem cells by a competitive repopulation strategy.Proc Natl Acad Sci U S A. 1990; 87: 8736Crossref PubMed Scopus (348) Google Scholar] indicated that 15–20% of the steady-state adult stem cells are CD34+. The ontogenetic changes in the proportions of CD34+ and CD34− stem cells are diagrammatically presented in Figure 1. Earlier, Osawa et al. [7Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell.Science. 1996; 273: 242Crossref PubMed Scopus (1699) Google Scholar] and we [16Sato T Laver J.H Ogawa M Reversible expression of CD34 by murine hematopoietic stem cells.Blood. 1999; 94: 2548PubMed Google Scholar] reported that all stem cells in the Lin−c-kit+Sca−1+ bone marrow cells of adult mice are CD34−. The discrepancy between these observations must be due to the enrichment protocol prior to cell sorting used in the earlier [7Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell.Science. 1996; 273: 242Crossref PubMed Scopus (1699) Google Scholar, 16Sato T Laver J.H Ogawa M Reversible expression of CD34 by murine hematopoietic stem cells.Blood. 1999; 94: 2548PubMed Google Scholar] studies. Some stem cells may express lineage-specific markers [23Szilvassy S.J Cory S Phenotypic and functional characterization of competitive long-term repopulating hematopoietic stem cells enriched from 5-fluorouracil–treated murine marrow.Blood. 1993; 81: 2310PubMed Google Scholar] and thus may be eliminated by the negative immunoaffinity method used for preparing the Lin− cells. It has also been demonstrated that administration of 5-FU to mice activates stem cells and downregulates c-kit expression [24Randall T.D Weissman I.L Phenotypic and functional changes induced at the clonal level in hematopoietic stem cells after 5-fluorourical treatment.Blood. 1997; 89: 3596PubMed Google Scholar]. A small population of the stem cells in steady-state bone marrow of adult mice must be cycling to generate committed progenitors. These stem cells may be CD34+ and possibly c-kit− and/or Lin+. If so, the stem cell enrichment protocol used by Osawa et al. [7Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell.Science. 1996; 273: 242Crossref PubMed Scopus (1699) Google Scholar] and us [16Sato T Laver J.H Ogawa M Reversible expression of CD34 by murine hematopoietic stem cells.Blood. 1999; 94: 2548PubMed Google Scholar] may have eliminated CD34+ adult stem cells before FACS sorting based on CD34 expression. Another long-held dogma is that human stem cells and primitive progenitors are CD34+CD38−/low [25Novielli E.M Ramirez M Civin C.I Biology of CD34+CD38− cells in lymphohematopoiesis.Leuk Lymphoma. 1998; 31: 285PubMed Google Scholar]. However, studies of the murine model documented clearly that both adult and fetal murine stem cells are CD38+ [26Randall T.D Lund F.E Howord M.C Weissman I.L Expression of murine CD38 defines a population of long-term reconstituting hematopoietic stem cells.Blood. 1996; 87: 4057PubMed Google Scholar, 27Dagher R.N Hiatt K Traycoff C Srour E.F Yoder M.C c-kit and CD38 are expressed by long-term reconstituting hematopoietic cells present in the murine yolk sac.Biol Blood Marrow Transplant. 1998; 4: 69Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar]. In order to solve the controversy surrounding the two species we again studied the effects of activation of stem cells on their expression of CD38. First, we confirmed the published finding that the majority of long-term engrafting cells from normal adult steady-state marrow are CD38+ [28Tajima F Deguchi T Laver J.H Zeng H Ogawa M Reciprocal expression of CD38 and CD34 by adult murine hematopoietic stem cells.Blood. 2001; 97: 261Crossref Scopus (63) Google Scholar]. Next, we determined that the majority of stem cells activated in vivo by injection of 5-FU or mobilized by G-CSF are CD38−. Stem cells that were activated in culture with IL-11 and steel factor were also CD38−. As reviewed already, expression of CD34 by adult stem cells is modulated by in vivo or in vitro activation. In order to determine if there is reciprocal expression of CD38 and CD34, we analyzed four populations of bone marrow cells from 5-FU–treated mice that were separated based on CD34 and CD38 expressions. No stem cell activities were seen in the CD38+CD34+ or CD38−CD34− cell populations. The majority of the stem cells were in the CD38−CD34+ fraction. Secondary transplantation experiments indicated that when the bone marrow reaches steady state, the majority of the stem cells become CD38+CD34−. In an earlier section I described that a minority population of CD34+ stem cells persist in the bone marrow of adult mice [22Ito T Tajima F Ogawa M Developmental changes of CD34 expression by murine hematopoietic stem cells.Exp Hematol. 2000; 28: 1269Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar]. These CD34+ stem cells in the steady-state bone marrow of adult mice proved to be CD38−. These experiments with adult murine stem cells clearly indicated reciprocal relationship of CD34 and CD38 expression [28Tajima F Deguchi T Laver J.H Zeng H Ogawa M Reciprocal expression of CD38 and CD34 by adult murine hematopoietic stem cells.Blood. 2001; 97: 261Crossref Scopus (63) Google Scholar]. These series of studies of murine stem cells may clarify most of the conflicting reports on the surface markers of stem cells. The studies of the total population of murine stem cells [22Ito T Tajima F Ogawa M Developmental changes of CD34 expression by murine hematopoietic stem cells.Exp Hematol. 2000; 28: 1269Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar] clearly documented that the majority of the stem cells in the normal adult mice are CD34−, while a minority is in the CD34+ population. This agrees with the observations in the xenotransplantation studies that both CD34+ and CD34− populations of human cells are capable of long-term engraftment [13Zanjani E.D Almeida-Porada G Livingston A.G Flake A.W Ogawa M Human bone marrow CD34− cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells.Exp Hematol. 1998; 26: 353PubMed Google Scholar, 14Bhatia M Bonnet D Murdoch B Gan O.I Dick J.E A newly discovered class of human hematopoietic cells with SCID-repopulating activity.Nat Med. 1998; 4: 1038Crossref PubMed Scopus (523) Google Scholar]. The studies of activation of murine stem cells carried out in vivo by using 5-FU [16Sato T Laver J.H Ogawa M Reversible expression of CD34 by murine hematopoietic stem cells.Blood. 1999; 94: 2548PubMed Google Scholar] or G-CSF [19Tajima F Sato T Laver J.H Ogawa M CD34 expression by murine hematopoietic stem cells mobilized by G-CSF.Blood. 2000; 96: 1989PubMed Google Scholar] and in culture in the presence of IL-11 and SF [16Sato T Laver J.H Ogawa M Reversible expression of CD34 by murine hematopoietic stem cells.Blood. 1999; 94: 2548PubMed Google Scholar] clearly established the influence of activation on stem cell CD34 expression. Likewise, the effects of stem cell activation can explain the apparent interspecies differences in CD38 expression, i.e., adult human and murine stem cells are CD38− and CD38+, respectively. Randall et al. [26Randall T.D Lund F.E Howord M.C Weissman I.L Expression of murine CD38 defines a population of long-term reconstituting hematopoietic stem cells.Blood. 1996; 87: 4057PubMed Google Scholar] clearly demonstrated that stem cells in adult mice are CD38+ [26Randall T.D Lund F.E Howord M.C Weissman I.L Expression of murine CD38 defines a population of long-term reconstituting hematopoietic stem cells.Blood. 1996; 87: 4057PubMed Google Scholar]. However, the investigators studied highly purified populations of murine bone marrow cells such as Lin−Sca-1+c-kit+ population, which should contain only CD34− stem cells [16Sato T Laver J.H Ogawa M Reversible expression of CD34 by murine hematopoietic stem cells.Blood. 1999; 94: 2548PubMed Google Scholar]. We documented that activated CD34+ stem cells and the minority population of CD34+ stem cells in normal adult mice are CD38− [28Tajima F Deguchi T Laver J.H Zeng H Ogawa M Reciprocal expression of CD38 and CD34 by adult murine hematopoietic stem cells.Blood. 2001; 97: 261Crossref Scopus (63) Google Scholar]. This observation agrees with the CD38 expression by human stem cells because studies of CD38 expression by human stem cells are based on CD34+ human cells [25Novielli E.M Ramirez M Civin C.I Biology of CD34+CD38− cells in lymphohematopoiesis.Leuk Lymphoma. 1998; 31: 285PubMed Google Scholar]. Our murine studies of developmental changes in CD34 expression documented that the transition from CD34+ to CD34− stem cells takes place just before adulthood [22Ito T Tajima F Ogawa M Developmental changes of CD34 expression by murine hematopoietic stem cells.Exp Hematol. 2000; 28: 1269Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar]. This observation may explain the apparent inconsistencies among studies of murine stem cells. For example, Morel et al. [8Morel F Szilvassy S.J Travis M Chen B Galy A Primitive hematopoietic cells in murine bone marrow express the CD34 antigen.Blood. 1996; 88: 3774PubMed Google Scholar, 9Morel F Galy A Chen B Szilvassy S.J Equal distribution of competitive long-term repopulating stem cells in the CD34+ and CD34− fractions of Thy-1lowLin−/lowSca-1+ bone marrow cells.Exp Hematol. 1998; 26: 440PubMed Google Scholar] used 4- to 6-week-old mice and noted that either all [8Morel F Szilvassy S.J Travis M Chen B Galy A Primitive hematopoietic cells in murine bone marrow express the CD34 antigen.Blood. 1996; 88: 3774PubMed Google Scholar] or a significant number [9Morel F Galy A Chen B Szilvassy S.J Equal distribution of competitive long-term repopulating stem cells in the CD34+ and CD34− fractions of Thy-1lowLin−/lowSca-1+ bone marrow cells.Exp Hematol. 1998; 26: 440PubMed Google Scholar] of stem cells are CD34+. We observed that CD34− stem cells emerge after 5 weeks of age [22Ito T Tajima F Ogawa M Developmental changes of CD34 expression by murine hematopoietic stem cells.Exp Hematol. 2000; 28: 1269Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar]. Murine hematopoiesis has been a good model for human hematopoiesis and basic principles regulating murine stem cells appear to apply to human stem cells. Based on this premise, a number of predictions may be made about human stem cells. It is likely that a significant population of adult human stem cells do not express CD34. Studies using the sheep/human xenotranplantation technique appear to support this premise [29Zanjani E.D Almeida-Porada G Livingston A.G Harada N Zeng H.Q Ogawa M Reversible expression of CD34 by adult human bone marrow long-term engrafting cells in vivo.Blood. 2001; 98: 72aGoogle Scholar]. Although there are numerous reports on transplantation of CD34+ human bone marrow cells, almost all studies were based on cases of autologous transplantation. While CD34 selection provides an excellent method of purification of human stem cells, I believe it may inadvertently discard a sizable population of stem cells when applied to marrow cells from normal adults. It is very likely that the mobilized human stem cells are CD34+. The literature of clinical transplantation of allogeneic CD34+ cells clearly is supportive of this concept [2Bensinger W.I Buckner C.D Shannon-Dorcy K et al.Transplantation of allogeneic CD34+ peripheral blood stem cells in patients with advanced hematologic malignancy.Blood. 1996; 88: 4132PubMed Google Scholar, 3Link H Arseniev L Bahre O Kadar J.G Diedrich H Poliwanda H Transplantation of allogeneic CD34+ blood cells.Blood. 1996; 87: 4903PubMed Google Scholar, 4Hassan H.T Zeller W Stockschlader M Kruger W Hoffknecht M.M Zander A.R Comparison between bone marrow and G-CSF–mobilized peripheral blood allografts undergoing clinical scale CD34+ cell selection.Stem Cells. 1996; 14: 419Crossref PubMed Scopus (33) Google Scholar, 5Kawano Y Takaue Y Watanabe A et al.Partially mismatched pediatric transplants with allogeneic CD34+ blood cells from a related donor.Blood. 1998; 92: 3123PubMed Google Scholar]. The murine studies justify CD34 selection in enrichment of human mobilized blood cells for stem cells and provide experimental basis for the current clinical practice that the total number of CD34+ cells in mobilized blood is used as a predictor for engraftment. Stem cells in the pathological or non–steady-state bone marrow may vary in CD34 expression. For example, in the bone marrow of patients recovering from chemotherapy or radiotherapy, significant populations of the stem cells may be activated and express CD34. CD34 selection of the stem cells in these patients may have to be approached with caution. The murine model predicts that the majority of stem cells in human cord blood and the bone marrow of young children are CD34+. Precisely at what age the transition from CD34+ to CD34− stem cells takes place in man and what is the ratio of CD34+ to CD34− stem cells in adult humans remain to be tested directly on human stem cells. Finally, the reciprocal pattern of CD34 and CD38 expression by adult murine stem cells may also apply to adult human stem cells. If so, CD34+CD38− G-CSF–mobilized blood cells should be an excellent source for human hematopoietic stem cells. Unknown is the state of CD38 expression by the steady-state and activated stem cells of the juveniles. Studies are under way to examine this question using the murine model. This work was supported by NIH grants RO1-DK54197 and PO1-CA78582 and by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs. The author acknowledges the assistance of Anne G. Livingston in the preparation of the manuscript.