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Hematopoietic stem cell (HSC) heterogeneity and hierarchy are a current topic of interest, having major implications for clinical HSC transplantation and basic research on human HSCs. It was long believed that the most primitive HSCs in mammals, including mice and humans, were CD34 antigen positive (CD34+). However, 2 decades ago, it was reported that murine long-term multilineage reconstituting HSCs were lineage marker negative (Lin–, i.e., c-kit+Sca-1+CD34low/–), known as CD34low/– KSL cells. In contrast, human CD34– HSCs, a counterpart of murine CD34low/– KSL cells, were hard to identify for a long time mainly because of their rarity. We previously identified very primitive human cord blood (CB)-derived CD34– severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection method and proposed the new concept that CD34– SRCs (HSCs) reside at the apex of the human HSC hierarchy. Through a series of studies, we identified two positive/enrichment markers: CD133 and GPI-80. The combination of these two markers enabled the development of an ultrahigh-resolution purification method for CD34– as well as CD34+ HSCs and the successful purification of both HSCs at the single-cell level. Cell population purity is a crucial prerequisite for reliable biological and molecular analyses. Clonal analyses of highly purified human CD34– HSCs have revealed their potent megakaryocyte/erythrocyte differentiation potential. Based on these observations, we propose a revised road map for the commitment of human CB-derived CD34– HSCs. This review updates the current understanding of the stem cell nature of human CB-derived primitive CD34– as well as CD34+ HSCs.

Umbilical cord blood transplantation (UCBT) is often associated with delayed neutrophil and platelet recovery. Engraftment failure is another major obstacle. Several factors influence these serious complications, including the numbers of total nucleated cells (TNCs) and CD34+ cells which have been used as reliable factors for selecting UCB units for transplantation. However, whether both factors are reliable indices of the hematopoietic stem cell (HSC) activity of UCB units remains unknown. To evaluate the quality of UCB units, we quantified the actual number of transplantable CD34+CD133+ HSCs (tHSCs) residing in UCB units. The number of tHSCs was not correlated with the numbers of TNCs or CD34+ cells. These results strongly suggest that neither factor reflects the numbers of tHSCs residing in UCB units. To validate the significance of the number of tHSCs, further analysis is required to determine whether the number of tHSCs residing in UCB units is useful as a new indicator for the quality assessment of UCB units.

Murine bone marrow (BM)-derived very small embryonic-like stem cells (BM VSELs), defined by a lineage-negative (Lin(-)), CD45-negative (CD45(-)), Sca-1-positive (Sca-1(+)) immunophenotype, were previously reported as postnatal pluripotent stem cells (SCs). We developed a highly efficient method for isolating Lin(-)CD45(-)Sca-1(+) small cells using enzymatic treatment of murine bone. We designated these cells as bone-derived VSELs (BD VSELs). The incidences of BM VSELs in the BM-derived nucleated cells and that of BD VSELs in bone-derived nucleated cells were 0.002% and 0.15%, respectively. These BD VSELs expressed a variety of hematopoietic stem cell (HSC), mesenchymal stem cell (MSC), and endothelial cell markers. The gene expression profile of the BD VSELs was clearly distinct from those of HSCs, MSCs, and ES cells. In the steady state, the BD VSELs proliferated slowly, however, the number of BD VSELs significantly increased in the bone after acute liver injury. Moreover, green fluorescent protein-mouse derived BD VSELs transplanted via tail vein injection after acute liver injury were detected in the liver parenchyma of recipient mice. Immunohistological analyses suggested that these BD VSELs might transdifferentiate into hepatocytes. This study demonstrated that the majority of the Lin(-)CD45(-)Sca-1(+) VSEL phenotypic cells reside in the bone rather than the BM. However, the immunophenotype and the gene expression profile of BD VSELs were clearly different from those of other types of SCs, including BM VSELs, MSCs, HSCs, and ES cells. Further studies will therefore be required to elucidate their cellular and/or SC characteristics and the potential relationship between BD VSELs and BM VSELs.

We and others have reported that human hematopoietic stem cells (HSCs) are also present in the CD34-negative (CD34−) fraction of human cord blood (CB). Here, we examined the hematopoietic engraftment potential of 13 or 18 lineage-negative (13Lin− or 18Lin−) CD34+/− cells from human CB in mice and sheep. Both 13Lin− and 18Lin− CD34+ cells efficiently engrafted in mice irrespective of transplantation route, be it by tail-vein injection (TVI) or by intra-bone marrow injection (IBMI). These cells also engrafted in sheep after in utero fetal intra-hepatic injection (IHI). In contrast, neither 13Lin− nor 18Lin− CD34− cells engrafted in either mice or sheep when transplanted by regular routes (i.e., TVI and fetal IHI, respectively), although both 13Lin− and 18Lin− CD34− cells engrafted in mice when transplanted by IBMI and exhibited multilineage reconstitution ability. Thus, the homing ability of CD34− HSCs is significantly more limited than that of CD34+ HSCs. As for 18Lin−, CD34− HSCs are characterized by low expression of the tetraspanin CD9, which promotes homing, and high expression of the peptidase CD26, which inhibits homing. This unique expression pattern homing-related molecules on CD34− HSCs could thus explain in part their reduced ability to home to the BM niche.

The identification of human CD34-negative (CD34(-)) hematopoietic stem cells (HSCs) provides a new concept for the hierarchy in the human HSC compartment. Previous studies demonstrated that CD34(-) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) are a distinct class of primitive HSCs in comparison to the well-characterized CD34(+)CD38(-) SRCs. However, the purification level of rare CD34(-) SRCs in 18 lineage marker-negative (Lin(-)) CD34(-) cells (1/1000) is still very low compared with that of CD34(+)CD38(-) SRCs (1/40). As in the mouse, it will be necessary to identify useful positive markers for a high degree of purification of rare human CD34(-) SRCs. Using 18Lin(-)CD34(-) cells, we analyzed the expression of candidate positive markers by flow cytometric analysis. We finally identified CD133 as a reliable positive marker of human CB-derived CD34(-) SRCs and succeeded in highly purifying primitive human CD34(-) HSCs. The limiting dilution analysis demonstrated that the incidence of CD34(-) SRCs in 18Lin(-)CD34(-)CD133(+) cells was 1/142, which is the highest level of purification of these unique CD34(-) HSCs to date. Furthermore, CD133 expression clearly segregated the SRC activities of 18Lin(-)CD34(-) cells, as well as 18Lin(-)CD34(+) cells, in their positive fractions, indicating its functional significance as a common cell surface maker to isolate effectively both CD34(+) and CD34(-) SRCs.

To the editor: We identified very primitive CD34-negative (CD34−) severe combined immunodeficiency–repopulating cells (SRCs) in human cord blood (CB) using the intrabone marrow injection method[1][1] and provided a new concept for the hierarchy in the human hematopoietic stem cell (HSC)

We studied the effect of human flt3/flk2 ligand (FL) on the proliferation and differentiation of purified CD34 + blood progenitors which express different levels of c-kit protein in clonal cell culture in comparison with that of stem cell factor (SCF). FL alone did not support significant colony formation. However, FL significantly enhanced neutrophil colony (CFU-G) formation in the presence of granulocyte-colony stimulating factor (G-CSF) by peripheral blood (PB)-derived CD34 + c-kit - cells which contained a large number of CFU-G. In addition, FL could synergistically increase the number of CFU-G supported by a combination of interleukin (IL)-3 and G-CSF, as did SCF. As we reported previously, SCF showed a significant burst-promoting activity (BPA). In contrast, FL did not exhibit any BPA on PB-derived CD34 + c-kit high cells in which erythroid-burst (BFU-E) was highly enriched. However, FL could synergize with IL-3 or GM-CSF in support of erythrocyte-containing mixed (E-Mix) colony by PB-derived CD34 + c-kit high or low cells in the presence of Epo. Replating of E-Mix colonies derived from CD34 + c-kit high cells supported by IL-3+Epo+SCF yielded more secondary colonies than those supported by IL-3+Epo or IL-3+Epo+FL. When PB-derived CD34 + c-kit low cells which represent a more immature population than CD34 + c-kit high cells were used as the target, number of secondary colonies supported by IL-3+Epo, IL-3+Epo+SCF or IL-3+Epo+FL was comparable. However, the number of lineages expressed in the secondary culture was significantly larger in the primary culture containing IL-3+Epo+FL than in that containing IL-3+Epo. These results suggest that FL not only acts on neutrophilic progenitors, but also on more immature multipotential progenitors.

Human cord blood-derived primitive CD34-negative hematopoietic stem cells (HSCs) are myeloid-biased long-term repopulating HSCs

Using the intra-bone marrow injection (IBMI) technique, we recently identified human cord blood-derived CD34- severe combined immunodeficiency (SCID)-repopulating cells (SRCs) with extensive lymphomyeloid reconstituting ability. In this study, we further investigated the hematopoietic stem cell (HSC) characteristics of these cells in terms of proliferative and migratory potentials. The absolute numbers of CD45+ and CD34+ cells generated by 1 CD34- SRC are significantly higher than those generated by 1 CD34+ SRC. It is interesting that CD34- SRCs have significantly higher migratory and proliferative abilities than CD34+ SRCs. Moreover, only 2 CD34- SRCs transplanted to primary recipients consistently showed secondary reconstituting capacity. This finding suggested the more homogenous nature of CD34- SRCs than that of the population of CD34+ SRCs. These results provided further evidence that CD34- SRCs are functionally different from CD34+ SRCs and that they are a distinct class of primitive HSCs.

We have been investigating the hematopoietic stem cell (HSC) activity of peripheral blood-derived CD34(+) cells selected by two different laboratory immunomagnetic beads systems (MiniMACS and Isolex 50). In this study, the quality of purified CD34(+) cells was directly compared using clonal cell culture, a cobblestone area-forming cell (CAFC) assay, and an in vivo severe combined immunodeficiency (SCID)-repopulating cell (SRC) assay. It was found that CD34(+) cells selected by these two immunomagnetic methods showed a reduced yield of colony-forming cells and CAFCs compared with cells enriched by the StemSep device (a negative selection method). However, these CD34(+) cells still showed significant SRC activity, including multilineage lymphomyeloid reconstitution. The percentage of human CD45(+) cells in murine bone marrow after transplanting 5 x 10(5) CD34(+) cells selected by the Isolex 50 was significantly lower than after transplanting cells selected by the MiniMACS or the StemSep. Our findings clearly demonstrated that CD34(+) cells selected by the MiniMACS system had superior HSC functions, including SRC activity, compared with cells separated by the Isolex 50 system. More detailed functional analysis of immunomagnetically separated CD34(+) cells may provide useful knowledge for basic research on HSCs as well as for clinical HSC transplantation.

The Wilms' tumour gene, WT1, is expressed at high levels in leukaemia cells and plays an important role in leukaemogenesis. WT1 is also expressed in human normal CD34+ bone marrow (BM) cells at about 100 times lower levels than in leukaemia cells. To identify and characterize WT1-expressing cells in CD34+ BM cells, they were sorted into single cells and analysed for WT1 expression using two kinds of single-cell reverse transcriptase polymerase chain reaction (RT-PCR) methods. Using the semiquantitative single-cell polyA-PCR + sequence-specific (SS)-PCR method, WT1 expression was detected in four (1.3%) out of 319 CD34+ BM single cells. To confirm the above results, a single-cell nested sequence-specific (NSS)-RT-PCR method that was less quantitative but more sensitive than the polyA-PCR + SS-PCR method was also performed, and WT1 expression was detected in 15 (1.1%) out of 1315 CD34+ BM single cells. In total, WT1 expression was found in 19 (1.2%) out of 1634 CD34+ BM single cells. No significant differences in the frequencies of WT1-expressing cells were found between CD34+CD38- and CD34+CD38+ BM single cells. Furthermore, WT1-expressing CD34+ BM single cells expressed WT1 at levels similar to those in K562 leukaemia single cells. Analysis of lineage-specific and cell cycle gene expression in WT1-expressing CD34+ BM single cells showed that the WT1 gene could be expressed in both uncommitted, dormant CD34+CD38- and lineage-committed, proliferating CD34+CD38+ BM cells. Our results could indicate that these WT1-expressing CD34+ BM cells were normal counterparts of leukaemia cells.

We assessed the effect of signalling through CXCR4 on the proliferation and differentiation of human megakaryocytic progenitor cells (CFU-Meg) in the presence or absence of stem cell factor (SCF) and/or thrombopoietin (TPO), using peripheral blood-derived CD34(+)IL-6R(-) cells as a target. TPO alone induced a significant number of CFU-Meg colonies. Although stromal cell-derived factor-1 (SDF-1) or SCF alone did not support CFU-Meg colony formation, these factors had a synergistic effect on CFU-Meg colony formation in the presence of TPO. The combination of SDF-1, SCF and TPO induced twice as many CFU-Meg colonies as TPO alone. To investigate the mechanism of this synergistic action, we examined the effects of various protein kinase inhibitors on CFU-Meg colony formation. LY294002 and GF109203X (inhibitors of PI3-K and PKC respectively) completely or partially inhibited this synergistic action. In contrast, a MEK inhibitor (PD98059) did not inhibit CFU-Meg colony formation. It significantly increased the higher ploidy classes (16N to 64N) of megakaryocytes supported by TPO, TPO + SCF, TPO + SDF-1, and TPO + SCF + SDF-1, whereas it abolished the effect of SDF-1 on the increase of higher ploidy classes of megakaryocytes supported by TPO. These results suggest that MAPK may negatively or positively regulate the nuclear maturation of megakaryocytes, known as endomitosis. In the presence of PD98059, proplatelet formation (PPF) was significantly augmented, suggesting that the MAPK pathway may also inhibit the initiation of PPF. In conclusion, simultaneous activation of three signals through c-mpl, c-kit and CXCR4 can induce the in vitro proliferation and differentiation of CFU-Meg, and SDF-1 is a potentiator of human megakaryocytopoiesis.

Background. Over the past two decades, the fields of umbilical cord blood (UCB) banking and UCB transplantation (UCBT) have been established. Over 600,000 UCB units have been stored for UCBT world-wide, and over 30,000 UCBTs have already been performed. In Japan in particular, the number of UCBTs has rapidly increased, and over 11,000 UCBTs have been performed thus far. However, UCBT is often associated with delayed neutrophil and platelet recovery compared with bone marrow transplantation. Furthermore, a major obstacle to overcome in UCBT is engraftment failure, which can often be a life-threatening complication. There are several factors influencing graft failure, including the numbers of total nucleated cells (TNCs) and CD34+ cells, the degree of HLA disparity, the presence or absence of anti-HLA antibody, and inappropriate immune suppression before UCBT. Among these factors, the numbers of TNCs and CD34+ cells have been used as dependable factors for selecting UCB units for transplantation. However, whether or not both factors can be used as reliable indices of the HSC activity of UCB units remains to be confirmed. Aim . In order to evaluate the quality of UCB units for UCBT, we quantified the actual numbers of transplantable CD34+CD133+ HSCs residing in UCB units. Materials and Methods. We previously identified CD133 as a positive marker of human CB-derived CD34+/- SCID-repopulating cells (SRCs) (Leukemia 28:1308,2014). CB-derived 18Lin-CD34+/-CD133+/- cells were sorted by FACS, and the HSC capacities of these four fractions of cells were extensively investigated. All of the mice that received 18Lin-CD34+/-CD133+ cells showed primary and secondary human CD45+ cell repopulations. However, none that received 18Lin-CD34+/-CD133- cells showed any human cell repopulation. These results showed that the CD133 expression clearly segregated SRC activities into the positive fractions. Furthermore, a limiting dilution analysis demonstrated that the frequencies of SRCs in the 18Lin-CD34+/-CD133+ fraction were 1/99 and 1/142, respectively. Based on these data, we developed a new method for accurately measuring the numbers of transplantable CD34+/- CD133+ SRCs residing in UCB units. Results. We measured the numbers of transplantable CD34+CD133+ SRCs (HSCs) residing in 449 UCB units. The numbers of TNCs ranged from 8.5 to 34.2 x 108 (median, 14.9 x 108) and the numbers of CD34+ cells ranged from 2.3 to 18.3 x 106 (median, 4.1 x 106). Surprisingly, the numbers of CD34+CD133+ HSCs showed an unexpectedly wide range from 144 to 61,866 (median, 3,994). These results clearly demonstrated that the HSC number varied widely among UCB units. Next, we statistically analyzed the HSC numbers of each UCB unit using quartile deviation. The data were divided into three groups based on the HSC quartiles: the lower group (A) including the data under the first quartile, the higher group (C) including data above the third quartile, and the middle group (B) including everything in between. The Pearson and the Spearman correlation coefficients between the HSC number and TNCs or CD34+ cells were calculated for each group. As shown in the Table, the respective numbers of TNCs and CD34+ cells ranged from (A) 8.5 to 28.1 x 108 (median, 14.2 x 108) and 2.3 to 13.6 x 106 (median, 3.6 x 106), (B) 9.0 to 34.2 x 108 (median, 14.7 x 108) and 2.5 to 12.4 x 106 (median, 4.0 x 106), and (C) 10.1 to 29.1 x 108 (median, 15.7 x 108) and 2.6 to 18.3 x 106 (median, 5.5 x 106). In contrast, the numbers of CD34+CD133+ HSCs ranged widely: (A) 144 to 2,088 (median, 1,259), (B) 2,092 to 6,897 (median, 3,994), and (C) 7,032 to 61,866 (median, 10,533). Except for (C), the Pearson and the Spearman correlation coefficients were close to zero, showing no correlation between the numbers of CD34+CD133+ HSCs and the numbers of TNCs and CD34+ cells. Conclusion. These results strongly suggest that the numbers of TNCs and CD34+ cells do not represent the numbers of CD34+CD133+ HSCs residing in the UCB units. A prospective clinical observation study is now ongoing to investigate the correlation of the numbers of CD34+CD133+ HSCs and the clinical indexes, including neutrophil and platelet recovery as well as graft failure after UCBT. We anticipate that the numbers of CD34+CD133+ HSCs residing in UCB units may be useful as a new indicator for quality assurance of UCB units. Disclosures No relevant conflicts of interest to declare.

Background. We identified very primitive human cord blood (CB)-derived CD34-negative (CD34-) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection (IBMI) method (Blood 2003:101;2924). Recently, we identified CD133 as a positive marker of human CB-derived CD34+/- SRCs. Limiting dilution analyses (LDAs) demonstrated that the frequencies of SRCs in the 18Lin-CD34+/-CD133+ fractions were 1/100 and 1/140, respectively (Leukemia 2014:28;1308). We then identified glycosylphosphatidylinositol-anchored surface protein (GPI-80) as a useful enrichment marker of human CB-derived CD34+/- SRCs and succeeded in highly purifying primitive human CD34+/- SRCs to the level of 1/20 cells. Aim. We attempted to purify CD34+/- SRCs to the single-cell level using CD133 and GPI-80 in order to precisely characterize the CD34- HSCs in comparison to CD34+ HSCs. Materials and Methods. We first developed an ultra-high-resolution purification method using two positive/enrichment markers for CD34+/- SRCs, including CD133 and GPI-80. We sorted 18Lin-CD34+CD38-CD133+GPI-80+ (34+38-133+80+)and 18Lin-CD34- CD133+GPI-80+ (34-133+80+)cells by FACS. Thereafter, these two fractions of cells were transplanted by the IBMI technique into NOG/NSG mice to investigate their long-term repopulating capacities. Finally, we performed single-cell transplantations. We then analyzed the gene expression profiles of single 34+38-133+80+ and 34-133+80+ cells using a BioMark System (Fluidigm). In parallel, we performed microarray experiments to analyze their gene expression profiles. Results. Approximately 14% of 34+38-133+ cells and 8% of 34-133+ cells expressed GPI-80. These highly purified cells showed very immature blast-like morphologies. These two fractions of cells were then transplanted into NOG/NSG mice by IBMI. We performed primary and secondary transplantations for up to 40 weeks. All of the mice that received 200 34+38-133+80+ (n=25)and 34-133+80+ (n=23) cells were repopulated with human CD45+ cells, including CD34+, CD19+ and CD33+ cells. Almost all of the secondary transplanted mice showed comparable human CD45+ cell reconstitution with multi-lineage differentiation. An LDA demonstrated that the frequencies of CD34+/- SRCs in the 34+38-133+80+ and 34-133+80+ cells were 1/5 and 1/8, respectively. Interestingly, 13.2% (7/53) of the recipient NSG mice that received single 34+38-133+80+ cells displayed distinct human CD45+ cell reconstitution (0.5%-44.2%) with multi-lineage human cell repopulation at 20 weeks after transplantation. In contrast, 6.6% (5/76) of the recipient NSG mice that received single 34-133+80+ cells displayed distinct human CD45+ cell reconstitution (0.6%-30.1%) with multi-lineage human cell repopulation at 20 weeks after transplantation. Both groups of reconstituted mice showed secondary reconstitution with multi-lineage differentiation. These results indicated that individual 34+38-133+80+ and 34-133+80+ cells extensively self-renew. We then analyzed the gene expression profiles of these two types of SRCs at the single-cell level. A principle component analysis showed that the gene expression profiles of individual CD34+ and CD34- SRCs were clearly different. Both SRCs expressed high levels of HSC maintenance genes, including RUNX1 and BMI1. Very interestingly, CD34+ SRCs expressed a high level of IFITM1, IKZF1 and ETV6. In contrast, CD34- SRCs expressed higher levels of EZH2 than CD34+ SRCs. Furthermore, a gene set enrichment analysis demonstrated that 34+38-133+80+ cells expressed higher levels of genes related to cell adhesion, chemokine signaling, and trans-endothelial migration, whereas 34-133+80+ cells expressed higher levels of genes related to developmental maturation. Very interestingly, 34-133+80+ cells expressed lower levels of interferon signal-related genes, including STAT1, IFTM1, IFTM3, Ddx58, IFI44, CCL5 and CXCL10, than 34+38-133+80+ cells. These results suggest that different mechanisms control HSC self-renewal and maintenance, as well as epigenetic regulation in these two types of CD34+/- SRCs. Conclusion. Wedeveloped an ultra-high-resolution purification method using two markers for CD34+/- SRCs, including CD133 and GPI-80. This precise single cell-based analysis allows us to map CD34- SRCs (HSCs) at the apex of the human HSC hierarchy. Disclosures No relevant conflicts of interest to declare.

The significance of interleukin 6 receptor (IL-6R) expression by cord blood (CB)- and peripheral blood (PB)-derived primitive haematopoietic progenitors was investigated. IL-6R was preferentially expressed by PB-derived myeloid progenitors. Most PB-derived erythroid bursts (BFU-E) and mixed colony-forming cells (CFU-Mix) did not express this receptor. However, CB-derived primitive progenitor cells possessed multipotentiality, irrespective of IL-6R expression. Interestingly, the long-term culture-initiating cell (LTC-IC) population was enriched in PB-derived CD34+ IL-6R+ cells, but the extended LTC-IC (ELTC-IC) population, which represents a less mature class of haematopoietic progenitors, seemed to be equally distributed in the IL-6R+ and IL-6R− cell populations. In contrast, the number of LTC-ICs and ELTC-ICs was similar in CB-derived CD34+ IL-6R+ or IL-6R− cells. It is noteworthy that the number of LTC-ICs and ELTC-ICs in CB-derived CD34+ cells was markedly higher than that in PB-derived CD34+ cells regardless of IL-6R expression. Telomerase activity was consistently lower in PB-derived CD34+ IL-6R− cells than in CD34+ IL-6R+ cells. In contrast, telomerase activity was similar in CB-derived CD34+ IL-6R+ or IL-6R− cells. The pattern of telomerase induction upon cytokine stimulation differed between CB- and PB-derived CD34+ IL-6R+ or IL-6R− cells. However, overall telomerase activity per dish was well correlated with the proliferative potential of both cell populations, suggesting that induction of telomerase plays an important role in the escape from replicative senescence of primitive haematopoietic progenitors. Collectively, these results suggest that CB-derived primitive progenitors are less mature than PB-derived progenitors and that the expression of IL-6R by primitive haematopoietic progenitors may have different implications for PB- and CB-derived CD34+ cells.

We studied the functional characteristics of subpopulations of cord blood-derived CD34+ cells expressing different levels of CD38 and c-kit antigens, using clonal cell culture and long-term culture with allogeneic bone marrow stromal cells or the MS-5 murine stromal cell line to assay long-term culture-initiating cells (LTC-IC) in each subpopulation. To investigate the capacity for replication, proliferation, and differentiation of each subpopulation of CD34+ cells, we also studied the correlation between LTC-IC and telomerase activity. After 5 weeks of coculture, LTC-IC accounted for one out of 32 CD34+CD38- cells and one out of 33 CD34+c-kit- cells. In contrast, the frequency of LTC-IC was low in their antigen-positive counterparts (one per 84 CD34+CD38+ cells, one per 90 CD34+c-kit(low) cells, and very low among CD34+c-kit(high) cells). It was noteworthy that some LTC-IC derived from CD34+CD38- as well as CD34+c-kit- cells generated colony-forming cells (CFCs) after up to 9 weeks of coculture. Telomerase activity was consistently low in CD34+CD38- and CD34+c-kit- cells compared to CD38+ or c-kit(high or low) cells, suggesting that CD34+CD38- or c-kit- cells are likely to be more quiescent. These results suggest that the CD34+CD38- and CD34+c-kit- cell populations are primitive stem/progenitor cells, and that the telomerase activity of these cells correlates with their proliferative capacity as well as their stage of differentiation.

We studied the interaction of interleukin (IL)-4 and other burst-promoting activity (BPA) factors, such as IL-3, granulocyte/macrophage colony-stimulating factor (GM-CSF), IL-9 and stem cell factor (SCF), on erythroid burst-forming unit (BFU-E) and erythrocyte-containing mixed (CFU-Mix) colony formation in serum-free culture. IL-4 alone did not support mixed colony formation in the presence of erythropoietin (Epo). However, IL-4 showed weak but significant BPA when peripheral blood (PB)-derived CD34 + c-kit low cells were used as the target population. The BPA of IL-4 was much weaker than that of IL-3, which exerted the most potent activity, as previously reported. When CD34 + c-kit high cells were used as the target, four factors known to have BPA, IL-3, GM-CSF, IL-9 and SCF, could express BPA. In contrast, IL-4 alone failed to support erythroid burst formation. Interestingly, IL-4 showed a remarkable enhancing effect with SCF in promoting the development of erythroid burst and erythrocyte-containing mixed colonies from CD34 + c-kit low and CD34+c-kit high cells. Delayed addition of SCF+Epo or IL-4+Epo to the cultures initiated with either IL-4 or SCF alone clearly demonstrated that SCF was a survival factor for both BFU-E and CFU-Mix progenitors. In contrast, the survival effect of IL-4 was much weaker than that of SCF, and appeared to be more important for progenitors derived from CD34 + c-kit low cells than for those derived from CD34 + c-kit high cells. It was recently reported that CD34 + c-kit low cells represent a more primitive population than CD34 + c-kit high cells. Taken together, these results suggest that IL-4 helps to recruit primitive progenitor cells in the presence of SCF.

To clarify whether the expression of the WT1 gene in leukemic cells is aberrant or merely reflects that in normal counterparts, the expression levels of the WT1 gene were quantitated for normal hematopoietic progenitor cells. Bone marrow (BM) and umbilical cord blood (CB) cells were fluorescence-activated cell sorting (FACS)-sorted into CD34+ and CD34− cell populations, and the CD34+ cells into nine subsets (CD34+CD33−, CD34+CD33+, CD34+CD38−, CD34+CD38+, CD34+HLA-DR−, CD34+HLA-DR+, CD34+c-kithigh, CD34+c-kitlow, and CD34+c-kit−) according to the expression levels of CD34, CD33, CD38, HLA-DR, and c-kit. Moreover, acute myeloid leukemic cells were also FACS-sorted into four populations (CD34+CD33−, CD34+CD33+, CD34− CD33+, and CD34− CD33−). FACS-sorted normal hematopoietic progenitor and leukemic cells and FACS-unsorted leukemic cells were examined for the WT1 expression by quantitative reverse transcriptase-polymerase chain reaction. The WT1 expression in the CD34+ and CD34− cell populations and in the nine CD34+ subsets of BM and CB was at either very low (1.0 to 2.4 × 10−2) or undetectable (

Background. We previously identified very primitive human cord blood (CB)-derived CD34-negative (CD34-) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection (IBMI) method (Blood 2003:101;2924). A series of our studies suggests that the CD34- SRCs that we identified are a distinct class of primitive hematopoietic stem cells (HSCs), which show myeloid-biased long-term repopulating capacity, suggesting that they are at the apex of the human HSC hierarchy (Blood Cancer J 2015:5,e290). We recently developed a high-resolution purification method for primitive CD34- SRCs using 18 lineage (Lin)-specific antibodies, which can enrich CD34- SRC at the 1/1,000 level (Exp Hematol 2011:39;203). We then identified CD133 as a positive marker of human CB-derived CD34+/- SRCs. Moreover, limiting dilution analyses (LDAs) demonstrated that the frequencies of SRCs in the 18Lin- CD34+/- CD133+ fractions were 1/100 and 1/140, respectively (Leukemia 2014:28;1308). Since we aim to purify the CD34- SRCs (HSCs) at the single cell level, it was necessary to identify other specific positive markers for CD34- SRCs. We extensively analyzed the expression of candidate positive markers in the 18Lin- CD34- cell population by FACS. Finally, we identified glycosylphosphatidyl-inoshitol-anchored surface protein (GPI-80) as a useful marker of human CB-derived CD34+/- SRCs and succeeded in highly purifying primitive human CD34+/- SRCs to the level of 1/20 cells. Aim. We herein attempted to purify CD34+/- SRCs to the single cell level in order to precisely characterize the CD34- SRCs (HSCs) in the human HSC hierarchy. Materials and Methods. We first developed an ultra-high resolution purification method using two reliable markers for CD34+/- SRCs, including CD133 and GPI-80. Namely, we sorted 18Lin- CD34+ CD38- CD133+ GPI-80+ (34+ 38- 133+ 80+)and 18Lin- CD34- CD133+ GPI-80+ (34- 133+ 80+)cells by FACS. Thereafter, these two fractions of cells were transplanted by the IBMI technique into NOD/Shi-scid/IL-2Rγcnull (NOG) mice to investigate their long-term repopulating capacities. We ultimately performed single cell transplantations and analyzed their human hematopoietic cell reconstitution for up to 20 weeks. Finally, we analyzed their gene expression profiles, including the key genes for the self-renewal and maintenance of HSCs of single 34+ 38- 133+ 80+ and 34- 133+ 80+ cells using a BioMark System (Fluidigm). Results. Approximately 15% of the 34+ 38- 133+ and 34- 133+ cells expressed GPI-80. These highly purified cells showed very immature blast-like morphologies. These two fractions of cells were then transplanted into NOG mice by IBMI. We performed primary and secondary transplantations for up to 40 weeks. In the results, all of the mice received 200 34+ 38- 133+ 80+ (n=25)and 34- 133+ 80+ (n=23) cells were repopulated with human CD45+ cells (Mean % of human CD45+ cells, 47% vs. 35%), including CD34+, CD19+ and CD33+ cells. Most of the secondary transplanted mice were also repopulated with human CD45+ cells with multi-lineage reconstitution (Mean % of human CD45+ cells, 0.4% vs. 10%). An LDA is currently underway; however, the frequencies of CD34+/- SRCs in the 34+ 38- 133+ 80+ and 34- 133+ 80+ cells are estimated to be 1/5-1/10. Interestingly, significant numbers of the recipient mice that received single cells displayed multi-lineage human cell repopulation at 20 weeks after transplantation. In order to provide an independent line of evidence for characterizing our highly purified CD34+/- SRCs, we analyzed the gene expression profiles of these two types of SRCs at the single cell level. The principle component analysis clearly demonstrated that the gene expression profiles of individual CD34+ and CD34- SRCs were clearly different. Both SRCs expressed high levels of HSC maintenance genes, including RUNX1, TAL1, BMI1 and MYBC. Very interestingly, CD34+ SRCs expressed a high level of ETV6. In contrast, CD34- SRCs expressed higher levels of EZH2 and RING1. These results suggest that different mechanisms control HSC self-renewal and maintenance, as well as epigenetic regulation in these two SRCs. Conclusion. Wedeveloped an ultra-high resolution purification method using two markers for CD34+/- SRCs, including CD133 and GPI-80. The precise single cell-based analysis allows us to map CD34- SRCs (HSCs) at the apex of the human HSC hierarchy. Disclosures No relevant conflicts of interest to declare.

Background. We identified very primitive CD34-negative (CD34-) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) in human cord blood (CB) using the intra-bone marrow injection (IBMI) method (Blood 2003:101;2924). These CD34- SRCs possess myeloid-biased differentiation potential, which suggests that they are a distinct class of primitive hematopoietic stem cell (HSC) and that they reside at the apex of the human HSC hierarchy (Blood Cancer J 2015:5;e290). We recently developed high-resolution purification methods for CD34- SRCs using 18 Lineage (18Lin)-specific antibodies, which can enrich CD34- SRC at the 1/1,000 level in 18Lin- CD34- fractions (Exp Hematol 2011:39:203). In addition, we previously identified CD133 as a positive marker for CD34- SRCs as well as for CD34+ SRCs (Leukemia 2014:28;1308). The results showed that CD34+/- SRCs were enriched to approximately 1/100 and 1/140 in 18Lin- CD34+/- CD133+ fractions, respectively. Aim. In order to further elucidate the details of the characteristics of human CD34+/- HSCs, we aimed to identify additional positive markers for the high-level purification of CB-derived CD34+/- SRCs. Materials and Methods. First, weextensively analyzed the candidate positive markers, including cell adhesion molecules and homing receptors that are expressed on 18Lin- CD34+ CD38- and 18Lin- CD34- cells by multicolor FACS. Finally, we discovered that glycosylphosphatidylinositol-anchored protein GPI-80, which has recently been reported as a marker for human fetal liver hematopoietic stem/progenitor cells (HSPCs) (Cell Stem Cell 2015:16;80), was also expressed on human full-term CB-derived 18Lin- CD34+ CD38- and 18Lin- CD34- cells. Next, we sorted 18Lin- CD34+ CD38- GPI-80+/- and 18Lin- CD34- GPI-80+/- cells from human CB. The HSPC characteristics of the 18Lin- CD34+ CD38- GPI-80+/- and 18Lin- CD34- GPI-80+/- cells were assessed as follows: (1) the in vitro maintenance/production capacities of CD34+ and CD34+ CD38- cells were examined in co-cultures with mesenchymal stroma cells (MSCs) established from human bone marrow-derived CD45- Lin- CD271+ SSEA-4+ cells (Stem Cells 2015:33;1554); (2) an SRC assay was performed using NOD/Shi-scid/IL-2Rγcnull (NOG) mice; (3) limiting dilution analyses (LDAs) were performed to determine the SRC frequencies in these four fractions of cells. Results. In the CB-derived 18Lin- CD34+ CD38- and 18Lin- CD34- fractions, 10.1% and 14.4% of cells expressed GPI-80, respectively. The 18Lin- CD34+ CD38- GPI-80+/- and 18Lin- CD34- GPI-80+/- cells were then co-cultured with human MSCs for 7 days in the presence of SCF and TPO. As a result, the 18Lin- CD34+ CD38- GPI-80+ cells maintained significantly higher percentages of CD34+ (86.4%) and CD34+ CD38- cells (24.8%) in comparison to 18Lin- CD34+ CD38- GPI-80- cells (78.7% and 14.3%, respectively). However, 18Lin- CD34- GPI-80+/- cells produced comparable levels of CD34+ (50.3% and 50.8%) and CD34+ CD38- cells (4.4% and 5.3%). These four fractions of cells were then transplanted into NOG mice using the IBMI method. All of these four fractions of cells showed long-term repopulating SRC activities with multi-lineage differentiation potential in mouse bone marrow. However, LDAs demonstrated that the frequencies of SRC in the 18Lin- CD34+ CD38- GPI-80+/- and 18Lin- CD34- GPI-80+/- fractions were 1/21, 1/35 and 1/28, 1/874, respectively. These data clearly demonstrate that both CD34+/- SRCs are enriched in GPI-80+ fractions. Surprisingly, a number of mice received a limited number of 18Lin- CD34+ CD38- GPI-80+ (2 cells) and 18Lin- CD34- GPI-80+ cells (10 cells), and thus also showed multi-lineage long-term human hematopoietic cell repopulation. Conclusion. These observations clearly demonstrated that GPI-80 defines CB-derived human primitive HSCs. Furthermore, these results indicate that GPI-80 is a useful marker for the high-level purification of human CB-derived CD34+/- SRCs (HSCs). Disclosures No relevant conflicts of interest to declare.

Interleukin-4/B-cell stimulatory factor-1 (IL-4/BSF-1) is a unique cytokine which may have multiple regulatory functions in vitro and in vivo. It has been shown to produce diverse effects on hematopoietic progenitors and can act on the proliferation and differentiation of committed as well as primitive hematopoietic progenitors. It acts synergistically with G-CSF to support neutrophil colony formation. In contrast, it inhibits IL-3-dependent colony formation and macrophage colony formation supported by IL-3 plus M-CSF, GM-CSF, or by M-CSF alone. It also suppresses pure and mixed megakaryocyte colony formation supported by IL-3 in the presence of Erythropoietin (EPO). IL-4, however, supports multipotential blast cell colony formation. And there are apparent differences between the functions of IL-4 and IL-3. IL-4 is able to exert its hematopoietic actions directly and indirectly. For example, it enhances the release of GM-CSF or G-CSF from T-lymphocytes and TNF-alpha from monocytes. Since IL-4 receptors have been shown to be expressed on bone marrow stromal cells as well as hematopoietic cells, it may also act in the bone marrow microenvironment. These results suggest that IL-4 is an intermediate-acting, lineage-non-specific factor just like IL-3 or GM-CSF. Complex interactions between many cytokines including IL-4 may act in the regulation of normal as well as pathological hematopoiesis.

We studied the effects of recombinant human interleukin-4 (rhIL-4) on megakaryocyte colony formation from enriched hematopoietic progenitors. IL-4 strongly inhibited pure and mixed megakaryocyte colony formation in a dose-dependent manner. Formation of erythroid bursts, eosinophil colonies, and erythrocyte-containing mixed colonies was not affected by the addition of IL-4 as reported previously (Sonoda Y, et al; Blood 75:1615, 1990). Delayed addition experiments suggested that IL-4 acts on an early stage of proliferation of megakaryocyte progenitors. Neutralizing antibodies (antisera) prepared against transforming growth factor beta, tumor necrosis factor alpha, interferon alpha (IFN alpha), and IFN gamma did not affect the inhibitory effects of IL-4 on pure and mixed megakaryocyte colony formation. In addition, the inhibitory effects of IL-4 was also seen in serum-free cultures and in cultures containing highly enriched CD34+, HLA-DR+ cells as a target population. These results indicate that IL-4 may function as one of the negative regulators in human megakaryocytopoiesis in vitro.

We have investigated the biological activities of recombinant human interleukin-9 (IL-9) on enriched hematopoietic progenitors, alone or in combination with other cytokines, including Epo, G-CSF, IL-3, and GM-CSF, under serum-containing and serum-free cultures. IL-9 alone did not support colony formation. However, IL-9 plus Epo induced erythroid burst (BFU-E) formation derived from peripheral blood (PB) progenitors. Delayed addition experiments demonstrated that a part of bone marrow (BM) derived BFU-E, which seems to be immature, only responded to IL-9 and formed erythroid bursts. The burst-promoting activity (BPA) of IL-9 was confirmed using neutralizing aIL-3, aGM-CSF, and aIL-9 antisera and serum-free culture. IL-9 supported a part of BFU-E population that respond to IL-3, which was almost identical to the number of BFU-E supported by GM-CSF. IL-9 had no additive effect on erythroid and mixed colony formation supported by IL-3. In contrast, IL-9 showed an additive effect on erythroid burst formation supported by GM-CSF in serum-free culture. These data suggest that IL-9 and GM-CSF act on distinct IL-3-responsive BFU-E population. In addition, delayed addition experiment clearly demonstrated that IL-9 supports survival and the early stage of proliferation of BFU-E. These results led us to propose that IL-9 possibly acts as a BPA and selectively supports a subpopulation of early class of BFU-E that respond to IL-3. © 1992 Wiley-Liss, Inc.

Stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4 are the key regulatory molecules of hematopoietic stem cell (HSC) migration and engraftment to the bone marrow (BM) microenvironment. However, the significance of the ligand-receptor complex on HSC in steady-state BM is not clear. There is currently a lack of information as to how CXCR4 is expressed on HSCs. We herein demonstrate that c-kit(+)Sca-1(+)Lineage(-) (KSL) cells freshly isolated from BM expressed very low to undetectable levels of CXCR4. Two hours of incubation at 37 degrees C quickly up-modulated the receptor expression on KSL cells. Protein synthesis was not required for this early stage up-regulation, thus suggesting the emergence of intracellularly pooled receptors to the cell surface. However, protein synthesis was involved at the later stage of up-regulation. The up-regulated CXCR4 was functional, as evidenced by the fact that the incubated KSL cells more efficiently migrated to the SDF-1 gradient in vitro. Therefore, although KSL cells are able to express functional CXCR4, the receptors are only marginally expressed in the steady-state BM microenvironment. These observations therefore indicate the limited role of the SDF-1-CXCR4 axis on HSC functionality in a static BM environment.

The TEL/ARG oncogene is formed by t(1;12)(q25;p13) reciprocal translocation and is associated with human leukemia. We have previously demonstrated that the expression of TEL/ARG in Ba/F3 cells results in prolonged viability and hyper-responsiveness to IL-3. To determine the molecular mechanisms, a series of mutants of TEL/ARG were generated, and each cDNA was expressed in Ba/F3 or CHO cells. The PNT domain in TEL and K317 in ARG were essential for both signaling and biological effects. The SH3 domain in ARG was required for hyper-responsiveness to IL-3, but not for prolonged viability. The opposite was true for the SH2 domain in ARG. Mutation of Y314 in TEL, a putative GRB2-binding site, led to reduced viability, and loss of hyper-responsiveness to IL-3. All biological functions were profoundly impaired with deletion of the C-terminus in ARG, despite maintaining high levels of its kinase activity. When expressed in CHO cells, wild-type TEL/ARG induced the formation of fillopodia, in a fashion dependent on the C-terminal portion and intact kinase activity. Thus, these results suggest several critical domains within TEL/ARG necessary for function, and indicate that the signaling pathways necessary for viability, growth factor hyper-responsiveness and cytoskeletal reorganization are likely to be separate.

The TEL/ARG oncogene associated with acute myeloid leukemia is formed by the t(l;12)(q25;pl3) reciprocal transloca-tion, which fuses part of the TEL gene to the tyrosinc kinasc, c-ARG. In an effort to determine the biological effects and investigate signaling of the TEL/ARG fusion protein, multiple sublines of Ba/F3 cells were generated in which a TEL/ARG complementary DNA was expressed under the control of a tetracycline-inducible promoter. Treatment of these cells with doxycycline, a tetracycline analogue, rapidly induced expression of the TEL/ARG protein. TEL/ARG was heavily phospho-rylated on tyrosine residues and was also found to rapidly induce tyrosine phosphorylation of multiple cellular proteins, including rasGAP, CBL, STATS, PI3K, SHP2, Dok, and SHC. The Ba/F3-tet-TEL/ARG cells remained interleukin (IL)-3 dependent without doxycycline but with doxycycline displayed a marked reduction in cell death in the absence of IL-3. TEL/ ARG cells also displayed an enhanced proliferative response to IL-3 and to insulin-like growth factor 1. At least in Ba/F3 cells, although the growth rate was much lower compared to that with IL-3, TEL/ARG appeared to induce some cell proliferation as an immediate consequence. Nonetheless, the hyperresponsiveness to growth factors reported here is more likely to contribute to the pathogenesis of leukemia.

Primitive hematopoietic progenitor cells such as severe combined immunodeficiency- repopulating cells and long-term culture-initiating cells are enriched in CD34+CD38- cells derived from various stem cell sources. In this study, to elucidate the features of such primitive cells at the molecular level, we tried to isolate genes that were preferentially expressed in umbilical cord blood (CB)-derived CD34+CD38- cells by subtractive hybridization. The gene for VPAC1 receptor, a receptor for the neuropeptide vasoactive intestinal peptide (VIP), was thereby isolated and it was shown that this gene was expressed in both CD34+CD38- and CD34+CD38+ CB cells and that the expression levels were higher in CD34+CD38- CB cells. Next, we assessed the effects of VIP on the proliferation of CD34+ CB cells using in vitro culture systems. In serum-free single-cell suspension culture, VIP enhanced clonal growth of CD34+ CB cells in synergy with FLT3 ligand (FL), stem cell factor (SCF), and thrombopoietin (TPO). In serum-free clonogenic assays, VIP promoted myeloid (colony-forming unit-granulocyte/macrophage (CFU-GM)) and mixed (CFU-Mix) colony formations. Furthermore, in Dexter-type long-term cultures, VIP increased colony-forming cells at week 5 of culture. These results suggest that VIP functions as a growth-promoting factor of CB-derived hematopoetic progenitor cells.

Objective This study investigated the effect of interleukin-9 (IL-9) on the proliferation and differentiation of human colony-forming unit megakaryocytic progenitor cells (CFU-Meg). Materials and Methods Peripheral blood-derived CD34 + IL-6R − cells were sorted and cultured in the presence of IL-9, erythropoietin (Epo), stem cell factor (SCF), and thrombopoietin (TPO) alone or in combination. The number of pure and mixed megakaryocyte colonies, the size of pure megakaryocyte colonies, the ploidy distribution of megakaryocytes, and proplatelet formation were investigated. Results Apart from TPO, no single factor could support CFU-Meg–derived colony formation, but each two-factor combination among IL-9, Epo, and SCF supported a few CFU-Meg colonies. Interestingly, the combination of Epo+SCF+IL-9 induced four to six times as many CFU-Meg colonies as any of the two-factor combinations. Neutralizing monoclonal antibodies (mAbs) for IL-9 receptor and c-kit completely abolished this synergistic effect. In contrast, addition of neutralizing anti–c-Mpl or anti-CXCR4 Abs did not influence colony formation, indicating that this synergistic effect was independent of TPO or SDF-1. Moreover, the endogenous production of TPO by cultured CD34 + IL-6R − cells in the presence of Epo+SCF+IL-9 was ruled out by reverse transcriptase polymerase chain reaction for TPO mRNA. Interestingly, the combination of TPO, Epo, SCF, and IL-9 supported the largest number of pure and mixed megakaryocyte colonies, suggesting that this combination of cytokines might recruit primitive megakaryocytic as well as multipotential progenitors. This combination also potently enhanced proplatelet formation compared with TPO alone or a combination of Epo, SCF, and IL-9. Conclusion This study demonstrated for the first time that human IL-9 can potentiate human megakaryocytopoiesis in the presence of Epo and/or SCF.

Precise analysis of human CD34-negative (CD34(-)) hematopoietic stem cells (HSCs) has been hindered by the lack of a simple and reliable assay system of these rare cells. Here, we successfully identify human cord blood-derived CD34(-) severe combined immunodeficiency (SCID)- repopulating cells (SRCs) with extensive lymphoid and myeloid repopulating ability using the intra-bone marrow injection (IBMI) technique. Lineage-negative (Lin(-)) CD34(-) cells did not show SRC activity by conventional tail-vein injection, possibly due to their low levels of homing receptor expression and poor SDF-1/CXCR4- mediated homing abilities, while they clearly showed a high SRC activity by IBMI. They generated CD34(+) progenies not only in the injected left tibia but also in other bones following migration. Moreover, they showed slower differentiating and reconstituting kinetics than CD34(+) cells in vivo. These in vivo-generated CD34(+) cells showed a distinct SRC activity after secondary transplantation, clearly indicating the long-term human cell repopulating capacity of our identified CD34(-) SRCs in nonobese diabetic (NOD)/SCID mice. The unveiling of this novel class of primitive human CD34(-) SRCs by IBMI will provide a new concept of the hierarchy in the human HSC compartment and has important implications for clinical HSC transplantation as well as for basic research of HSC.

We examined the effect of recombinant human interleukin (IL)-11 alone or in combination with various colony-stimulating factors (CSFs), including IL-3, granulocyte/macrophage (GM)-CSF, granulocyte (G)-CSF, stem cell factor (SCF), flt3 ligand (FL), and thrombopoietin (TPO), on colony formation by leukemic progenitor cells (L-CFU) obtained from 33 patients with acute myelogenous leukemia (AML). Leukemic colony formation was found in approximately 70 to 80% of the patients in the presence of at least one of the above CSFs. Although IL-11 alone did not support L-CFU, the growth of these progenitors in the presence of other cytokines was enhanced by IL-11 in 16 out of 33 patients and it showed a synergistic action with G-CSF in 12 of them. This synergistic action occurred in seven out of nine M5 patients (French-American-British (FAB) classification). A single cell clone-sorting experiment clearly demonstrated that this synergistic effect was operative at the single progenitor cell level. The number of leukemic cells proliferating in the presence of G-CSF+IL-11 was significantly higher than in the presence of G-CSF alone, suggesting that IL-11 recruited dormant leukemic progenitors into the cell cycle. Flow cytometric analysis revealed that all types of AML blast cells (M0 approximately M6) ubiquitously expressed gp130, although the level of expression was significantly higher in M5 cells. In contrast, expression of the IL-11 receptor alpha chain (IL-11Ralpha) varied between FAB types. Blast cells obtained from M1, M3 and M5 patients showed higher levels of expression, with M5 cells showing the strongest expression. Interestingly, the leukemic progenitor cells for which proliferation was synergistically enhanced by IL-11 had significantly higher expression of both IL-11Ralpha and gp130. These results suggest that administration of IL-11 in vivo may stimulate the proliferation of leukemic progenitor cells, particularly M5 cells, in the presence of G-CSF, and that the responsiveness of L-CFU to IL-11 may be predicted by a simple receptor assay.

In this study, we examined a large number of patients to clarify the distribution and frequency of a recently described FLT3 tandem duplication among hematopoietic malignancies, including 112 acute myelocytic leukemia (AML), 55 acute lymphoblastic leukemia (ALL), 37 myelodysplastic syndrome (MDS), 20 chronic myelogenous leukemia (CML), 30 non-Hodgkin's lymphoma (NHL), 14 adult T cell leukemia, 15 chronic lymphocytic leukemia (CLL) and 38 multiple myeloma (MM). We also evaluated 71 cell lines derived from 11 AML, 31 ALL, two hairy cell leukemia, three acute unclassified leukemia, 10 CML, 12 NHL including six Burkitt's lymphoma, and two MM. Using genomic PCR of exon 11 coding for the juxtamembrane (JM) domain and first amino acids of the 5'-tyrosine kinase (TK) domain, this length mutation was found only in AML (22/112, 20%) and MDS (1/37). According to the FAB subclassification, they were 5/18 (28%) of M1, 4/29 (14%) of M2, 3/17 (18%) of M3, 6/24 (25%) of M4, 4/20 (20%) of M5 and 1/9 of refractory anemia with excess of blast in transformation. In the various cell lines examined, this abnormality was determined in only one derived from AML and never found in other hematological malignancies. The sequence analysis of the abnormal PCR products revealed that 23 of 24 showed internal tandem duplication with or without insertion of nucleotides. In one AML, insertion and deletion without duplication was determined. All 24 lengthened sequences were in-frame. Duplication takes place in the sequence coding for the JM domain and leaves the TK domain intact. In conclusion, we emphasize that the length mutation of FLT3 at JM/TK-I domains were restricted to AML and MDS. Since all these mutations resulted in in-frame, this abnormality might function for the proliferation of leukemic cells.

We studied the effects of stem cell factor (SCF) and flt3 ligand (FL) on the ex vivo expansion of human umbilical cord blood (CB)-derived CD34+ cells in combination with various cytokines, including interleukin (IL)-3, IL-6, IL-11, and c-Mpl ligand (thrombopoietin, TPO), in a short-term serum-free liquid suspension culture system. Among the two-factor combinations tested, SCF plus IL-3 most effectively expanded committed progenitor cells, including mixed colony-forming units (CFU-Mix). The expansion efficiency (EE) of FL for each progenitor was inferior to that of SCF in the presence of various cytokines, except TPO. IL-6 significantly increased the EE for granulocyte/macrophage colony-forming units (CFU-GM) obtained with SCF + IL-3 or FL + IL-3. Interestingly, TPO markedly augmented the EE for committed progenitors, including CFU-GM, erythroid burst-forming units (BFU-E), and CFU-Mix, in the presence of SCF + IL-3 or FL + IL-3. The combinations of SCF + IL-3 + TPO + IL-6 or IL-11 maximally stimulated the expansion of committed progenitors. The maximum EE for CFU-GM, BFU-E, and CFU-Mix was respectively 197-fold (day 14), 60-fold (day 7) and 51-fold (day 14). Other combinations of cytokines without IL-3 failed to expand effectively these committed progenitors. Our data demonstrate that it is possible to expand human CB-derived committed progenitors in vitro using SCF or FL with several other cytokines including TPO, and that IL-3 is the key cytokine promoting the expansion of human hematopoietic progenitors in the presence of SCF or FL.

Background We have previously identified very primitive human cord blood (CB)-derived CD34-negative (CD34-) severe combined immunodeficiency (SCID)- repopulating cells (SRCs) using the intra-bone marrow injection (IBMI) method (Blood 2003:101;2924). A series of our studies suggests that the identified CD34- SRCs are a distinct class of primitive hematopoietic stem cell (HSC) and that they are at the apex of human HSC hierarchy. Recently, we developed a high-resolution purification method for primitive CD34- SRCs using 18 lineage (Lin)-specific antibodies, which can enrich CD34- SRC at 1/1,000 level (Exp Hematol 2011: 39:203). In the present study, we tried to identify the positive marker of CD34- SRCs in order to further purify and characterize the CD34- SRCs (HSCs). Materials and Methods First, we extensively analyzed candidate positive markers, including known HSC markers and various adhesion molecules by FACS using highly purified CB-derived 18Lin-CD34+/- cells. Finally, we identified CD133 as a positive marker of human CB-derived CD34- SRCs. Then, CB-derived 18Lin- CD34+/-CD133+/- cells were sorted by FACS, and hematopoietic stem/progenitor cell (HSPC) capacities of these four fractions of cells were extensively investigated. HSPC capacities were evaluated using (1) colony-forming cell (CFC) assays, (2) measurement of maintenance/production of CD34+ cell capacities in co-cultures with human bone marrow-derived mesenchymal stromal cells (BM-MSCs) (Blood 2010:24:162), (3) SRC activities using NOG mice, (4) limiting dilution analyses (LDA) to determine the SRC frequency in the 18Lin-CD34-CD133+ fractions, and (5) comparison of gene expression profiles between 18Lin-CD34+/-CD133+/- cells by real-time RT-PCR. Results Seventy-five percent of 18Lin-CD34+ and 13.5% of 18Lin-CD34- cells highly expressed CD133. In the CFC assays, the plating efficiencies of 18Lin-CD34+CD133+, CD34+CD133-, CD34-CD133+ and CD34-CD133- cells were 57%, 65%, 39% and 19%, respectively. Interestingly, most of 18Lin-CD34-CD133+/- cells formed erythroid-bursts (71% and 73%) and erythro/megakaryocytes-containing mixed colonies (25% and 27%). On the contrary, they formed few granulocyte/macrophage colonies (4.2% and 0%). Then, we co-cultured these four fractions of cells with human BM-MSCs. One thousand of 18Lin-CD34+/-CD133+/- cells were seeded into each well and cells were co-cultured for 7 days in the presence of SCF+TPO+FL+IL-3+IL-6 +G-CSF. Both the 18Lin-CD34-CD133+/- cells produced CD34+ cells. However, the percentage and absolute number of CD34+ cells produced from 18Lin-CD34-CD133+ cells (31.7 % and 3.2 x 104 cells) were greater than those of 18Lin-CD34-CD133- cells (13.2 % and 0.4 x 104 cells). In addition, both the 18Lin-CD34- CD133+/- cells generated higher percentages (13.5 % and 11.5%) of CD41+ cells compared to those of the 18Lin- CD34+CD133+/- (1.8% and 4.2%) cells. Collectively, 18Lin-CD34+/-CD133+/- cells showed different in vitro lineage differentiation potentials. Then, these four fractions of cells were transplanted into NOG mice by IBMI. We performed primary and secondary transplantations for up to 36 weeks. In the results, all of the mice received 18Lin-CD34+CD133+ cells (n = 5) or 18Lin-CD34-CD133+ cells (n = 9) showed primary and secondary human CD45+ cell repopulations. However, neither 18Lin-CD34+CD133- cells nor 18Lin-CD34-CD133- cells showed human cell repopulations (n = 6 in each group). These results clearly demonstrated that the CD133 expression clearly segregated SRC activities in the 18Lin-CD34+/- cells. Moreover, LDA demonstrated that the frequency of SRCs in the 18Lin-CD34-CD133+ fraction was 1/142. Interestingly, HSC self-renewal maintenance genes, such as Notch1, HoxB4, HoxA9, and Bmi-1, were highly expressed in both 18Lin-CD34+/-CD133+ cells. Conclusion These results clearly demonstrated that CD133 is a positive marker of human CB-derived CD34- SRCs (HSCs). Furthermore, CD133 segregated SRC activities of 18Lin-CD34- as well as 18Lin-CD34+ cells in its positive fractions. More importantly, these findings suggest that number of CD133+ cells in cord blood units is a more appropriate marker to detect/predict HSC potentials in cord blood stem cell transplantation in comparison to currently used CD34+ cell numbers. Disclosures: No relevant conflicts of interest to declare.

Abstract 2345 Background: Ratajczak and his colleagues identified a unique population of very small embryonic-like (VSEL) stem cells in adult mouse bone marrow (BM) (Leukemia 2006:20;857). These VSELs are; 1) very small (∼4 μm); 2) express pluripotent stem cell markers, such as Oct4, Nanog, SSEA-1, and Rex-1; 3); responsive to a SDF-1 gradient; 4) possess large nuclei that contain euchromatin. It is very interesting to note that VSELs possess the potential to differentiate into 3 germ layers in vitro and in vivo, thereby contributing to tissue/organ regeneration. These VSELs were isolated as lineage-negative (Lin−), Sca-1-positive (Sca-1+), CD45-negative (CD45−) cells by FACS. However, the incidence of VSELs in BM-derived mononuclear cells is ∼0.01%. Therefore, it is difficult to isolate VSELs very effectively. This study describes our recently developed highly efficient method for isolating VSELs using enzymatic treatment of murine bone. Materials and Methods: Murine BM nucleated cells (BMNC) were isolated from BM flushed from the pairs of femurs and tibiae of 8 week-old C57BL/6 mice. Erythrocytes were removed using a hypotonic solution. Then the remaining bone tissues were thoroughly washed using PBS- with 2% FCS. These bone tissue specimens were crushed in a mortar and then incubated in cell dissociation buffer containing a-medium with 5% FCS supplemented with 1.5 mg/ml type I collagenase and 2 mg/ml dispase at 37°C for 1 hour. Next, the BMNCs and bone-derived nucleated cells (BDNCs) were stained with various monoclonal antibodies, including anti-lineages, anti-CD45, anti-Sca-1, anti-CXCR4, anti-CD133, and anti-PDGFRα, and then were used for subsequent FACS analyses. Results: The R1 gate was set on the FSC channel using 4 and 10 μm synthetic beads, based on the predicted very small size of VSELs. The VSELs were isolated from BMNCs and BDNCs by multicolor FACS, as a population of Lin−Sca-1+CD45− cells (Fig. 1A). The incidences of VSELs in the BMNCs and BDNCs were 0.001% and 0.1%, respectively. Therefore, the enzymatic treatment of bone tissues yielded about 100 times the efficiency for the isolation of VSELs (Fig. 1B). The bone-derived (BD) VSELs were small (< 5 μm) and possessed a relatively large nucleus surrounded by a narrow rim of cytoplasm. They expressed CD133, but not PDGFRα. However, they weakly expressed CXCR4. The gene expression profiles were analyzed using real time quantitative PCR (RQ-PCR) to evaluate the expression of ES cell markers (Oct4, Nanog, Rex1, Dppa3), HSC (KSL) markers (c-kit, Tal1, GATA2), and MSC markers (Nestin, Ang1, CXCL12, VE-Cadherin). Unexpectedly, BD VSELs expressed high levels of Nestin and Cadherin. However, they expressed weak levels of Oct4 and Nanog. The gene expression profile of the BD VSELs was clearly distinct from the well-defined populations of ES cells, KSL cells, and MSCs. Interestingly, the number of these BD VSELs significantly increased after the induction of liver injury by carbon tetrachloride administration. They were then most likely mobilized into the peripheral blood (PB). G-CSF did mobilize KSL cells into PB, as previously reported. However, G-CSF did not mobilize the BD VSELs. The effects of sRANKL on the mobilization of BD VSELs were examined in vivo. Interestingly, the number of BD VSELs significantly increased 2–3 days after the administration of sRANKL. However, the number of VSELs in PB did not increase. These results suggest that BD VSELs actively proliferated after liver injury and bone resorption. Conclusion: The present data suggest that the majority of the Lin−Sca-1+CD45− cells reside in the bone tissue. BD VSELs resemble BM-derived VSELs. However, a RQ-PCR analysis revealed that the gene expression profile of BD VSELs was different from those of the previously reported BM-derived VSELs. Further studies will therefore be required to elucidate their stem cell characteristics and the potential relationship between BD VSELs and BM-derived VSELs. Disclosures: No relevant conflicts of interest to declare.

We studied the synergistic effects of stem cell factor (SCF) and other burst-promoting activities (BPAs) such as interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or IL-9 on proliferation of human peripheral blood-derived highly purified progenitors. SCF, IL-3, GM-CSF, and IL-9 showed significant BPA when CD34+HLA-DR+ cells were used as the target population. IL-3 exerted the most potent BPA, and GM-CSF supported approximately 40% to 70% of the erythroid burst-forming units that are responsive to IL-3. SCF and IL-9 showed much weaker BPA than that of IL-3 or GM-CSF. Combinations of IL- 3 with other BPAs did not show synergistic actions supporting erythroid- burst formation. However, GM-CSF showed a significant additive effect with IL-9 or SCF. When CD34+c-kithigh cells were used as the target, SCF showed a much stronger BPA. Also, a distinct additive effect between SCF and IL-3 or GM-CSF on erythrocyte-containing mixed colony formation was observed. On the other hand, when CD34+c-kitlow cells were used as the target, SCF, IL-3, and GM-CSF could express BPA. In contrast, IL-9 alone failed to support erythroid-burst formation. Because CD34+c-kithigh cells weakly expressed CD34 antigen, these cells appeared to be more mature progenitors than CD34+c-kitlow cells. These results suggest that IL-9 acts on more mature progenitors than those of SCF, IL-3, or GM-CSF and that the primary target of SCF is multipotential progenitors at the very early stage of development.

Objective We have successfully identified human cord blood (CB)–derived CD34-negative (CD34 − ) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) with extensive lymphomyeloid repopulating ability using the intrabone marrow injection method. In our previous study, a limiting dilution analysis demonstrated the frequency of CD34 − SRCs in CB-derived 13lineage-negative (Lin − ) CD34 − cells to be approximately 1/25,000. In this study, we intended to develop a high-resolution purification method to obtain highly purified CD34 − SRCs. Materials and Methods The pooled CB-derived Lin − cells were stained with 13 reported Lin monoclonal antibodies (mAbs) and 5 more Lin mAb, against CD11b, CD33, CD66c, CD45RA, and CD127. Then 18Lin − CD34 high , 18Lin − CD34 − , and 13Lin − CD34 high CD38 − cells were sorted by fluorescence-activated cell sorting. Stem cell characteristics of these three fractions of cells were analyzed by in vitro cultures and in vivo repopulation assays for evaluation of this new purification method. Results A limiting dilution analysis demonstrated the frequency of CD34 − SRCs in these 18Lin − CD34 − cells to be approximately 1/1,000, which is associated with a seeding efficiency 25 times greater than the previous method. All primary recipient nonobese diabetic/Shi-scid/IL-2Rγc null mice that received transplants of only two CD34 − SRCs were highly engrafted with human lymphomyeloid cells at 24 weeks after primary transplantation and showed secondary multilineage repopulating abilities. Conclusions We succeeded to highly purify the CD34 − SRCs using 18Lin mAbs and the intrabone marrow injection technique. This newly developed high-resolution purification method is indispensable to precisely characterize a distinct class of primitive human CB-derived CD34 − hematopoietic stem cells.

Abstract 3852 (Background) The identification of human CD34-negative (CD34−) SCID-repopulating cells (SRCs) provide a new concept for the hierarchy in the human HSC compartment (Blood 101:2924, 2003). Recently, we succeeded to highly purify these CD34-SRCs using 18 lineage specific antibodies (Blood 114:336, 2009). It has been suggested that human hematopoietic stem cell (HSC)-supportive microenvironment exist in the bone marrow (BM), which play a pivotal role in the maintenance of self-renewal capacity and dormancy of primitive HSCs. It was reported that osteoblasts and vascular endothelial cells played an important role to organize HSC niches. However, whether mesenchymal stromal cells (MSCs) contribute to organize HSC niches or not is not clearly understood, because MSCs are heterogeneous population. Therefore, it is important to clarify their origin and functional characteristics. (Objectives) The aim of this study was to prospectively isolate/identify human BM-derived MSCs and investigate their functional characteristics including HSC-supportive abilities. (Results) First, human BM-derived Lin−CD45− cells were subdivided into 4 fractions according to their expression levels of CD271 and SSEA-4 by FACS. We succeeded to isolate 3 MSC lines from these 4 fractions, including CD271+/−SSEA-4+/− cells. Approximately 1 out of 6 CD271+SSEA-4+ (DP) cells could form MSC-derived colony. These DP cells-derived MSCs could differentiate into osteoblasts and chondrocytes, but could not differentiate into adipocytes. In contrast, CD271+SSEA-4− cells and CD271−SSEA-4− cells-derived MSCs could differentiate into three lineages. Then, we assessed CD34− SRC-supportive activity of these 3 MSC lines. First, certain numbers of 18Lin−CD34− cells were cocultured with 3 MSC lines for 1 week, respectively. Next recovered cells were transplanted into NOD/SCID mice by intra-bone marrow injection (IBMI) to investigate SCID-repopulating cell (SRC) activity. After 8 weeks, the highest CD45+ human cell engraftments (0.1 % to 32.4 %, median 8.6 %) were observed in mice received 18Lin−CD34− cells cocultued with DP cells-derived MSCs. As recently reported (Cell Stem Cell 1:635,2007), Lin−CD34+CD38−CD45RA−CD90+ cells contained most primitive human CD34+CD38− SRCs. Very interestingly, these Lin−CD34+CD38−CD45RA−CD90+ cells were generated from the above mentioned cocultures. In order to evaluate SRC activity of these Lin−CD34+CD38−CD45RA−CD90+ cells generated from 18Lin−CD34− cells in vitro, Lin−CD34+CD38−CD45RA−CD90+/− cells were sorted by FACS and then transplanted into NOD/SCID mice by IBMI. Eight weeks after transplantation, 8 out of 16 mice received Lin−CD34+CD38−CD45RA−CD90+ cells (400 to 3000 cells/mouse) were repopulated with human cells. In contrast, only 2 out of 16 mice received Lin−CD34+CD38− CD45RA−CD90− cells (1500 to 7000 cells/mouse) were repopulated. These results demonstrated that human CB-derived 18Lin−CD34− cells could generate very primitive CD34+CD38− SRCs in vitro. (Conclusion) These findings elucidate that human BM-derived DP cell-derived MSCs can support very primitive human CB-derived CD34− SRCs in vitro and suggest that these CD34− SRCs seem to be more immature than CD34+CD38− SRCs. These results provide a new concept of hierarchy in the human primitive HSC compartment. Disclosures: No relevant conflicts of interest to declare.

5 patients with refractory aplastic anemia (AA) received long-term administration (2-11 + months) of recombinant human G-CSF (rhG-CSF) in doses from 250-500 micrograms/body/day by intravenous infusion or 75-300 micrograms/body/d by subcutaneous injection. All 5 evaluable patients showed a substantial increase in absolute neutrophil count (ANC) with a recovery of myeloid components in the bone marrow after 1 to 2 months of treatment. Interestingly, 2 out of the 5 patients showed a dramatic improvement in severe anemia after 2 to 4 months of treatment accompanying a recovery of erythroid components in the bone marrow. In addition, there was no serious infection before or during therapy. Long-term administration of rhG-CSF was well tolerated because of its minimal toxicity. Clonal assay revealed a recovery of myeloid progenitors in all patients and a recovery of erythroid progenitors in 3 out of the 5 patients. These results suggest that long-term administration of rhG-CSF at least mobilizes residual myeloid as well as erythroid progenitor cells and induces a bilineage response in severe refractory AA.

Abstract 815 We have successfully identified human cord blood (CB)-derived CD34-negative (CD34−) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) with an extensive lymphoid and myeloid repopulating ability using the intra-bone marrow injection (IBMI) method (Blood 101:2924,2003). In that study, 13 lineage specific antibodies (Abs), including anti-CD2, CD3, CD4, CD7, CD10, CD14, CD16, CD19, CD20, CD24, CD41, CD56, and CD235a, were used to purify CD34− SRCs. A limiting dilution analysis demonstrated the frequency of CD34− SRCs in the 13 lineage-negative (Lin−) CD34− cells to be approximately 1/25,000. In this study, we added 5 more lineage specific Abs, including anti-CD11b, CD33, CD45RA, CD66c, and CD127, in order to highly purify CD34− SRCs. The 18 Lin−CD34− cells showed a homogeneously blast-like morphology, and their incidence in the CB-derived nucleated cells ranged from 0.0002 to 0.001%. The colony-forming capacity of these highly purified 18 Lin−CD34− cells was quite unique, since 50% of the total colony-forming cells (CFCs) were mixed colony-forming cells (CFU-Mix). In contrast, the 18 Lin−CD34+ cells formed myeloid, erythroid, and mixed colonies, however, only Disclosures: No relevant conflicts of interest to declare.

We administered recombinant human granulocyte colony-stimulating factor (rhG-CSF) intravenously for 2 weeks to 2 elderly patients with severe neutropenia. The absolute neutrophil count (ANC) recovered promptly after the initiation of rhG-CSF therapy and reached a peak (greater than 10 x 10(9)/l) on the 13th day. The ANC fell rapidly after rhG-CSF was discontinued, but it remained within the normal range after therapy. There were no side effects during the entire course of treatment. Therefore, rhG-CSF seems to be a most beneficial treatment in elderly patients with severe neutropenia.

Using the intra-bone marrow injection (IBMI) method, we have identified human cord blood (CB)-derived CD34-negative (CD34 − ) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) with multi-lineage repopulating ability (Blood101:2924,2003). Functional studies revealed that these CD34 − SRCs have different hematopoietic stem cell (HSC) characteristics from CD34 + SRCs. In order to further clarify the HSC characteristics of CD34 − SRCs, here we investigate the proliferative potential and redistribution kinetics of human CB-derived CD34 − SRCs, and compare them with those of CD34 + CD38 +/− SRCs using IBMI. First, we performed limiting dilution analyses and revealed that the incidence of CD34 + CD38 − SRCs in CB-derived Lin − CD34 + CD38 − cells was 1 out of 41 cells by IBMI. In contrast, the incidence of CD34 − SRCs in Lin − CD34 − cells was 1 out of 24,100, as we previously reported. Based on these data, we transplanted 200 to 5,000 Lin − CD34 + CD38 − cells (containing 5 to 120 SRCs), 15,000 to 50,000 Lin − CD34 + CD38 + cells (containing 10 to 30 SRCs), or 60,000 to 70,000 Lin − CD34 − cells (containing 3 SRCs) into primary recipient NOD/Shi-scid mice. After 5 weeks, all mice that received transplants of Lin − CD34 + CD38 +/− cells showed the human CD45 + cell repopulation in the other bones as well as the injected left tibiae. However, the human CD45 + cells were only detected in the injected left tibiae in mice that received transplants of Lin − CD34 − cells 5 weeks after the transplantation. In the mice that received transplants of 200 Lin − CD34 + CD38 − cells (containing 5 SRCs), the CD45 + CD34 + as well as CD45 + CD34 − cells were detected in both sites. In contrast, only CD45 + CD34 − cells were detected in the mice that received transplants of 70,000 Lin − CD34 − cells (3 SRCs). These results suggested that CD34 − SRCs might remain or slowly proliferate as CD34 − cells at the site of injection for at least 5 weeks. Next, we serially investigated the human CD45 + cell repopulation in the injected site and the other bones, separately. Very interestingly, CD34 + CD38 +/− SRCs began to migrate 2 weeks after the transplantation. The human cell repopulation in these mice was observed in other bones by 3 weeks after transplantation. Moreover, these CD34 + SRCs actively proliferated at both sites and produced CD34 + progenies. In contrast, CD34 − SRCs began to migrate 5 weeks after the transplantaion. Furthermore, these CD34 − SRCs showed significantly higher proliferative potential 8 weeks after transplantation than CD34 + SRCs and produced more CD34 + progenies not only at the site of injection, but also in the other bones. These results indicated that CD34 − SRC as well as CD34 + CD38 +/− SRCs could actively migrate from the injected site to the other bones. However, the time of initiation of migration was different between CD34 +/− SRCs. All these findings indicate that CD34 − SRCs show different proliferative potential and redistribution kinetics, and suggest that our identified CD34 − SRCs are distinct class of primitive HSCs in comparison with CD34 + CD38 +/− SRCs. We are now in the progress of clarifying whether the CD34 − SRCs migrate to other bones with the CD34 − immunophenotype or after their conversion (differentiation) to the CD34 + cells.

Recently, we have successfully identified human cord blood (CB)-derived CD34-negative (CD34−) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) with extensive lymphoid and myeloid repopulating ability using the intra-bone marrow injection (IBMI) method (Blood101:2924,2003). These CD34− SRCs could home into the BM niche only by IBMI, because they expressed lower levels of homing receptors including CXCR4. These CD34− SRCs did not express CD38 as well as c-kit. It is well documented that the tyrosine kinase receptors c-kit and flt3 are expressed and function in early mouse and human hematopoiesis. In murine model, it was reported that Lin−CD34−Sca−1+c-kit+flt3− cells supported long-term multilineage hematopoietic reconstitution. In contrast, Lin−CD34−Sca-1+c-kit+flt3+ cells are progenitors for the common lymphoid progenitor. More recently, these Lin−CD34−Sca-1+c-kit+flt3+ hematopoietic stem cells (HSCs) have been revealed to lack erythro-megakaryocytic potential. In this study, we have investigated the function of flt3 in our identified human CB-derived CD34− SRCs. First, we studied the SRC activity of CB-derived Lin-CD34+Flt3+/− or CD34−Flt3+/− cells using IBMI. Both CD34+FLT3+/− cells repopulated all 13 recipient mice. The level of human CD45+ cells in murine BMs received transplants of CD34+Flt3+ cells (29.3~90.8%, median 62.8%) was higher than those received transplants of CD34+Flt3− cells (9.8~45.1%, median 17.7%). On the other hand, only CD34−Flt3− cells repopulated all 7 recipient mice and the level of human CD45+ cells in murine BMs was 11.7~63.3% (median 37.9%). To further evaluate the long-term repopulating potential of these three populations, including CD34+Flt3+/− and CD34−Flt3− cells, BM cells obtained from each primary recipient mice were accessed for their SRC activities by secondary transplantation by IBMI. While CD34+Flt3+ cells did not show secondary repopulating activity, CD34+Flt3− cells could repopulate 83% (5/6) of secondary recipients. Moreover, CD34−Flt3− cells did repopulate all 5 secondary recipient mice with higher repopulating rate. Next, we cocultured CD34−Flt3−cells with the murine stromal cell line, HESS-5 in the presence of SCF, TPO, IL-3, IL-6, and G-CSF. After one week, significant numbers of CD34+Flt3− and CD34+Flt3+ cells were generated. Then we sorted these two populations, CD34+Flt3+/− cells, and tested their SRC activities by IBMI. Seven out of 10 and 5 out of 10 mice received CD34+Flt3+/− cells were repopulated with human cells, respectively. These results indicated that human CB-derived Lin−CD34−Flt3− cells produced CD34+Flt3− as well as CD34+Flt3+ SRCs in vitro. Our present study has demonstrated that human CB-drived CD34− SRCs do not express Flt3 tyrosine kinase receptor as did murine CD34− KSL cells. Based on these data, we propose that the immunophenotype of very primitive long-term repopulating human HSC is Lin−CD34−CD38−c-kit-Flt3−.

The Raf/MEK/ERK pathway is thought to be critical in mediating cell survival and proliferation by cytokine receptors. However, the exact contribution of Raf is complex and not well understood. A better understanding of Raf signaling is important because of the recent observation that B-Raf is frequently mutated in various human cancers. We have generated a new model system that activates Raf directly by linking the extracytoplasmic and transmembrane domains of the erythropoietin receptor (EPOR) with the catalytic domain of Raf (CR3). This synthetic oncogene in which dimerization can be controlled by an exogenous ligand, is fixed at the cellular membrane, while the endogenous Raf is normally activated by binding with Ras. The chimeric receptor (EPOR/CR3) was stably expressed in Ba/F3 cells which lack EPO receptors, and eight independent sublines were established. Although the lines remained dependent on IL-3 for proliferation, EPO treatment reduced the rate of cell death in the absence of IL-3. Also, EPO was synergistic with sub-optimal concentrations of IL-3 in inducing long-term cell proliferation, but did not augment proliferation of cells cultured with full concentrations of IL-3. EPO induced a rapid activation of ERK and also phosphorylation of endogenous Raf. It also induced tyrosine phosphorylation of several cellular proteins, estimated molecular masses of 150, 130, 110, 95, 60 and 42 kD. Many of the prominent targets of the wild type IL-3 or EPO receptors, such as Shc, Stat1, Stat3 and Stat5 were not tyrosine phosphorylated by the EPOR/CR3 receptor. The MEK1 inhibitor PD98059 reduced EPO-induced tyrosine phosphorylation, suggesting these substrates are downstream of MEK kinase. Interestingly, PD98059 also reduced the phosphorylation of endogenous Raf, indicating there is a positive feedback mechanism in Raf activation. We conclude that Raf can be activated by a mechanism that induces clustering at the cell membrane, and that this leads directly to activation of MEK and ERK. This is insufficient to induce proliferation by itself, but can add to sub-optimal growth signals from another receptor. This EPOR/CR3 system may serve as a useful model to evaluate the effects of signal transduction inhibitors for Raf as a target.

CD45 is a hematopoietic lineage-restricted antigen that is expressed on all hematopoietic cells except for some mature cell types. Cells expressing CD45 and CD34 but lacking CD38 and lineage antigens (CD45+CD34+CD38−Lin− cells) are well-documented hematopoietic stem cells (HSCs), and CD45+CD34−CD38−Lin− cells are probably less mature HSCs. In myelodysplastic syndromes (MDS), the malignant transformation site was reported to be committed myeloid progenitors and, more recently, the CD45+CD34+CD38−Lin− HSCs. In this study, we detected CD45−CD34−CD38−Lin− cells in the peripheral blood and bone marrow of MDS patients. Fluorescence in situ hybridization showed that CD45−CD34−CD38−Lin− cells had the same chromosomal aberration as the myeloblasts. In addition to CD45- and CD34-negativity, they lacked CD117 and CD133 expressions. Generally, MDS cells have extremely reduced hematopoietic potential compared with normal hematopoietic cells, but we documented the following in some cases. Freshly-isolated CD45−CD34−CD38−Lin− cells did not form any hematopoietic colonies but had long-term culture-initiating cell activity. When these cells were co-cultured with stroma cells, CD45−CD34−CD38−Lin− cells showed only weak potential for proliferation/differentiation, yet differentiated to CD34+ cells and then mature myeloid cells. This newly-identified cell population represents the most immature immunophenotype so far identified in the hematopoietic lineage and is involved in the malignant clone in MDS.