Search

Protection against subcutaneously inoculated OVA-expressing melanoma by OVA-peptide loaded iPS-pMC-DCs.

The use of dendritic cells (DC) to prime tumor-associated antigen-specific T-cell responses provides a promising approach to cancer immunotherapy. Embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) can differentiate into functional DCs, thus providing an unlimited source of DCs. However, the previously established methods of generating practical volumes of DCs from pluripotent stem cells (PSC) require a large number of PSCs at the start of the differentiation culture. In this study, we generated mouse proliferating myeloid cells (pMC) as a source of antigen-presenting cells (APC) using lentivirus-mediated transduction of the c-Myc gene into mouse PSC-derived myeloid cells. The pMCs could propagate almost indefinitely in a cytokine-dependent manner, while retaining their potential to differentiate into functional APCs. After treatment with IL4 plus GM-CSF, the pMCs showed impaired proliferation and differentiated into immature DC-like cells (pMC-DC) expressing low levels of major histocompatibility complex (MHC)-I, MHC-II, CD40, CD80, and CD86. In addition, exposure to maturation stimuli induced the production of TNFα and IL12p70, and enhanced the expression of MHC-II, CD40, and CD86, which is thus suggestive of typical DC maturation. Similar to bone marrow–derived DCs, they stimulated a primary mixed lymphocyte reaction. Furthermore, the in vivo transfer of pMC-DCs pulsed with H-2Kb-restricted OVA257-264 peptide primed OVA-specific cytotoxic T cells and elicited protection in mice against challenge with OVA-expressing melanoma. Overall, myeloid cells exhibiting cytokine-dependent proliferation and DC-like differentiation may be used to address issues associated with the preparation of DCs. Cancer Immunol Res; 3(6); 668–77. ©2015 AACR.

Supplementary Figure Legends

Supplementary Materials and Methods

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.

Summary It is generally thought that the proliferative capacity and differentiation potential of somatic stem cells, including mesenchymal stromal/stem cells (MSCs) and hematopoietic stem cells, decline with age. We investigated the effects of aging on human bone-derived MSCs expressing CD271 and SSEA-4 (double-positive MSCs [DPMSCs]). The percentages of DPMSCs in bone tissue decreased significantly with age. The DPMSCs from elderly patients (old DPMSCs) showed cellular senescence, which was evidenced by low growth potential, high senescence-associated β-galactosidase activity, and elevated p16 and p21 CDK inhibitor levels. Moreover, old DPMSCs showed weak osteogenic differentiation potential and less hematopoiesis-supporting activity in comparison with young DPMSCs. Interestingly, the addition of transforming growth factor β2 (TGF-β2) induced cellular senescence in young DPMSCs. With the exception of the adipogenic differentiation potential, all of the aging phenomena observed in old DPMSCs were reversed by the addition of anti-TGF-β antibodies. These results suggest that, in part, old DPMSCs accelerate cellular senescence through TGF-β signaling., Graphical Abstract, Highlights • The percentages of CD271+SSEA-4+ MSCs (DPMSCs) in bone tissue decline with age • DPMSCs may accelerate cellular senescence through TGF-β2 • TGF-β can change the balance of adipogenesis and osteogenesis of DPMSCs • The aging phenomena can possibly be reversed by anti-TGF-β antibodies, Sonoda et al. established bone-derived CD271+SSEA-4+ MSCs (DPMSCs) from young and elderly patients and demonstrated that DPMSCs may accelerate cellular senescence through TGF-β2 signaling. TGF-β can change the balance of adipogenesis and osteogenesis of DPMSCs. As a result, it may influence the impaired hematopoiesis observed in elderly patients. Interestingly, the aging phenomena can possibly be reversed by anti-TGF-β antibodies.

In the murine hematopoietic stem cell (HSC) compartment, thrombopoietin (THPO)/MPL (THPO receptor) signaling plays an important role in the maintenance of adult quiescent HSCs. However, the role of THPO/MPL signaling in the human primitive HSC compartment has not yet been elucidated. We have identified very primitive human cord blood (CB)-derived CD34–severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection method. In this study, we investigated the roles of the MPL expression in the human primitive HSC compartment. The SRC activities of the highly purified CB-derived 18Lin–CD34+/–MPL+/–cells were analyzed using NOG mice. In the primary recipient mice, nearly all mice that received CD34+/–MPL+/–cells were repopulated with human CD45+cells. Nearly all of these mice that received CD34+MPL+/–and CD34–MPL–cells showed a secondary repopulation. Interestingly, the secondary recipient mice that received CD34+/–MPL–cells showed a distinct tertiary repopulation. These results clearly indicate that the CD34+/–SRCs not expressing MPL sustain a long-term (LT) (>1 year) human cell repopulation in NOG mice. Moreover, CD34–SRCs generate CD34+CD38–CD90+SRCs in vitro and in vivo. These findings provide a new concept that CD34–MPL–SRCs reside at the apex of the human HSC hierarchy.

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.

Hematopoietic stem cell (HSC) biology is a current topic of interest having significant implications for clinical HSC transplantation and basic HSC research. It was long believed that the most primitive HSCs in mammals, including those in mice and humans, were CD34 antigen-positive (CD34

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

The use of dendritic cells (DC) to prime tumor-associated antigen-specific T-cell responses provides a promising approach to cancer immunotherapy. Embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) can differentiate into functional DCs, thus providing an unlimited source of DCs. However, the previously established methods of generating practical volumes of DCs from pluripotent stem cells (PSC) require a large number of PSCs at the start of the differentiation culture. In this study, we generated mouse proliferating myeloid cells (pMC) as a source of antigen-presenting cells (APC) using lentivirus-mediated transduction of the c-Myc gene into mouse PSC-derived myeloid cells. The pMCs could propagate almost indefinitely in a cytokine-dependent manner, while retaining their potential to differentiate into functional APCs. After treatment with IL4 plus GM-CSF, the pMCs showed impaired proliferation and differentiated into immature DC-like cells (pMC-DC) expressing low levels of major histocompatibility complex (MHC)-I, MHC-II, CD40, CD80, and CD86. In addition, exposure to maturation stimuli induced the production of TNFα and IL12p70, and enhanced the expression of MHC-II, CD40, and CD86, which is thus suggestive of typical DC maturation. Similar to bone marrow–derived DCs, they stimulated a primary mixed lymphocyte reaction. Furthermore, the in vivo transfer of pMC-DCs pulsed with H-2Kb-restricted OVA257-264 peptide primed OVA-specific cytotoxic T cells and elicited protection in mice against challenge with OVA-expressing melanoma. Overall, myeloid cells exhibiting cytokine-dependent proliferation and DC-like differentiation may be used to address issues associated with the preparation of DCs. Cancer Immunol Res; 3(6); 668–77. ©2015 AACR.

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)

Although c-kit is expressed highly on murine hematopoietic stem cells (HSCs) and essential for bone marrow (BM) hematopoiesis, the significance of the high level of expression of c-kit on HSCs was not well determined. We show here that CD150+CD48−Lineage−Sca-1+c-kit+ HSCs in adult BM are distributed within the range of roughly a 20-fold difference in the expression level of c-kit, and that c-kit density correlates with the cycling status of the HSC population. This predisposition is more evident in the BM of mice older than 30 weeks. The HSCs in G0 phase express a lower level of c-kit both on the cell surface and inside the cells, which cannot be explained by ligand receptor binding and internalization. It is more likely that the low level of c-kit expression is a unique property of HSCs in G0. Despite functional differences in the c-kit gradient, the HSCs are uniformly hypoxic and accessible to blood perfusion. Therefore, our data indicate the possibility that the hypoxic state of the HSCs is actively regulated, rather than them being passively hypoxic through a simple anatomical isolation from the circulation.

OECs (outgrowth endothelial cells), also known as late-EPCs (late-endothelial progenitor cells), have a high proliferation potential in addition to in vitro tube formation capability. In ischaemic animal models, injected OECs were integrated into regenerating blood vessels and improved neovascularization. Previous reports have demonstrated the expression of CXCL8 to be up-regulated in ischaemic tissues. It has also been documented that CXCL8 stimulates the angiogenic activity of mature ECs (endothelial cells). Therefore, it has been suggested that CXCL8 plays an important role in neovascularization in ischaemic tissues. However, it is still uncertain whether CXCL8 also stimulates the angiogenic activity of OECs. This study evaluated the effects of CXCL8 on the angiogenic activity of OECs in vitro. OECs were isolated from human UCB (umbilical cord blood)-derived mononuclear cells. Phenotypes of the OECs were assessed by flow cytometry, immunostaining, and real-time RT (reverse transcription)-PCR. The effects of CXCL8 on OECs were investigated by transwell migration assay and capillary tube formation assay on Matrigel. The OEC clones isolated from UCB expressed OEC phenotypes. In addition, CXCL8 receptors (CXCR1 and CXCR2) were expressed on these OEC clones. CXCL8 significantly stimulated the transwell migration and capillary tube formation of OECs. Neutralizing antibody against CXCR2, but not CXCR1, abolished a transwell migration of OECs induced by CXCL8, suggesting the involvement of CXCL8/CXCR2 axis in transwell migration. These results demonstrate that CXCL8 stimulates the angiogenic activity of UCB-derived OECs in vitro.

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.

Dietary restriction (DR) without malnutrition is widely acknowledged to prolong lifespan in laboratory animals. Evidence suggests that DR retards age-related decline in protein turnover of most organs. However, there has been no report about hepatic serum glycoprotein catabolism under DR. In the current study, we evaluate the hepatic uptake of asialoglycoprotein in ICR mice with DR by measuring the plasma clearance of technetium-99m galactosyl human serum albumin (Tc-GSA).The amount of food supplied to the restricted mice was 70% of that consumed by the mice fed ad libitum (AL). The regimen was initiated at the age of 7 weeks and terminated after the age of 44 weeks. The plasma clearance of Tc-GSA was measured at the age of 7 weeks, 14 weeks, 28 weeks, and 42 weeks.The restricted animals showed a marked decrease in their body and liver weight, and hepatic uptake of Tc-GSA per liver weight in the restricted mice was greater than that in the mice fed AL. On the other hand, the Tc-GSA plasma clearance in the mice fed AL was stable during the study period, and that in the restricted mice showed no change with age either, and those in the two groups were similar. In addition to the receptor function, there was no difference in the expression of mRNAs of the asialoglycoprotein receptor between the two groups. Serum concentrations of cholinesterase and hepatic mRNAs of glutamine synthetase in the restricted mice were higher than those in the mice fed AL. Serum levels of amino acids in the restricted mice were lower than those in the mice fed AL.The data presented here show that the DR did not affect the capacity of hepatic serum glycoprotein catabolism, whereas several protein metabolic pathways were affected.

We examined smoking prevalence among dentists in Hyogo, Japan, as smoking would influence their smoking cessation interventions to encourage their patients to stop smoking. In 2003, a self-administered questionnaire was mailed to all members of the Hyogo Dental Association (HDA) in Japan. Of the 1,133 members of the HDA, 327 were current smokers (28.9%). Smoking prevalence among HDA members was significantly higher than that among Japan Medical Association (JMA) members in 2004, as previously reported (16.2%) (p

Recently, we have identified 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). In contrast to murine CD34(-) Kit(+)Sca-1(+)Lineage(-) (KSL) cells, human CB-derived Lin(-)CD34(-) cells did not express detectable levels of c-kit by flow cytometry. In this study, we have investigated the function of flt3 in our identified human CB-derived CD34(-) SRCs. Both CD34(+)flt3(+/-) cells showed SRC activity. In the CD34(-) cell fraction, only CD34(-)flt3(-) cells showed distinct SRC activity by IBMI. Although CD34(+)flt3(+) cells showed a rather weak secondary repopulating activity, CD34(+)flt3(-) cells repopulated many more secondary recipient mice. However, CD34(-)flt3(-) cells repopulated all of the secondary recipients, and the repopulating rate was much higher. Next, we cocultured CD34(-)flt3(-) cells with the murine stromal cell line HESS-5. After 1 week, significant numbers of CD34(+)flt3(+/-) cells were generated, and they showed distinct SRC activity. These results indicated that CB-derived CD34(-)flt3(-) cells produced CD34(+)flt3(-) as well as CD34(+)flt3(+) SRCs in vitro. The present study has demonstrated for the first time that CB-derived CD34(-) SRCs, like murine CD34(-) KSL cells, do not express flt3. On the basis of these data, we propose that the immunophenotype of very primitive long-term repopulating human hematopoietic stem cells is Lin(-)CD34(-)c-kit(-)flt3(-). Disclosure of potential conflicts of interest is found at the end of this article.

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

Herbal teas have become popular as alternatives to caffeinated beverages during past two decades. However, toxicological studies of herbal teas have been limited and the safety of herbal teas thus remains unknown. We focused on the estrogenic activities of herbal teas since some of their ingredients are similar to those used in herbal remedies for menopause relief and therefore contain phytoestrogens. To investigate the potential estrogenic activity of extracts prepared from herbal tea mixtures commercially available and to provide useful information for the safety assessment of those products, we initially screened the estrogenic activity in extracts of 15 different herbal teas by an assay using recombinant yeast cells expressing the human estrogen receptor (YES). A distinct estrogenic activity was thus detected in the ethanolic extracts from four herbal tea mixtures. Licorice root was specified as a ingredient responsible for the estrogenic activity in those extracts. In contrast, the aqueous extracts of all herbal tea mixtures we tested exhibited distinct estrogenic activity in YES, thus suggesting the existence of various ingredients that contain estrogenic constituents extractable with water. Among them, the extract of peppermint tea exhibited the highest estrogenic activity. The estrogenic activity in extracts of herbal tea mixtures and specified ingredients were thereafter confirmed by a reporter assay system using transiently transfected HEK293 cells.

Metabolic rate is affected not solely by diet but also by environmental characteristics such as climate and seasonal changes in day length. In the present study, we conducted a controlled study in which we observed metabolic response to short-term energy restriction (ER).Thirty-two subjects were divided randomly into a slight ER group and a moderate ER group. The energy intake per day for slight ER vs moderate ER was 1462 kcal vs 1114 kcal. During the 4-day study periods, the same daily timetable, which consists of nutrition, exercise, sleeping and others, was imposed on both groups. The same environment was also provided to both groups.After the 4-day ER, significant decreases in body weight and basal metabolic rate (BMR) were shown in both groups. The decrease in body weight was 2% of the baseline level in both groups, and the decreases in the BMR were 6% of baseline levels in the slight ER group and 13% in the moderate ER group. The decrease in BMR in the moderate ER group was significantly larger than that in the slight ER group.In a controlled study of short-term ER, we observed a significant decrease in BMR. There was a positive association between the degree of ER and the reduction in BMR. Reductions in BMR were greater than those in body weight. It, thus, appears that the minimization of weight loss is due to dramatic decreases in BMR. This suggests the existence of metabolic resistance against ER.

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.

Hematopoietic stem cells (HSCs) are maintained in a specialized bone marrow (BM) niche, which consists of osteoblasts, endothelial cells, and a variety of mesenchymal stem/stromal cells (MSCs). However, precisely what types of MSCs support human HSCs in the BM remain to be elucidated because of their heterogeneity. In this study, we succeeded in prospectively isolating/establishing three types of MSCs from human BM-derived lineage- and CD45-negative cells, according to their cell surface expression of CD271 and stage-specific embryonic antigen (SSEA)−4. Among them, the MSCs established from the Lineage−CD45−CD271+SSEA-4+ fraction (DP MSC) could differentiate into osteoblasts and chondrocytes, but they lacked adipogenic differentiation potential. The DP MSCs expressed significantly higher levels of well-characterized HSC-supportive genes, including IGF-2, Wnt3a, Jagged1, TGFβ3, nestin, CXCL12, and Foxc1, compared with other MSCs. Interestingly, these osteo-chondrogenic DP MSCs possessed the ability to support cord blood-derived primitive human CD34-negative severe combined immunodeficiency-repopulating cells. The HSC-supportive actions of DP MSCs were partially carried out by soluble factors, including IGF-2, Wnt3a, and Jagged1. Moreover, contact between DP MSCs and CD34-positive (CD34+) as well as CD34-negative (CD34−) HSCs was important for the support/maintenance of the CD34+/− HSCs in vitro. These data suggest that DP MSCs might play an important role in the maintenance of human primitive HSCs in the BM niche. Therefore, the establishment of DP MSCs provides a new tool for the elucidation of the human HSC/niche interaction in vitro as well as in vivo. Stem Cells 2015;33:1554–1565

Based on the recent development of fluorescence-activated cell sorting (FACS) technology, murine hematopoietic stem cells (HSCs) can be purified at the single cell level. The immunophenotype of murine HSCs is CD34low/−c-kit+Sca-1+Lin− (CD34− KSL) cells. However, the characterization of primitive human HSCs has not been fully elucidated. The biology of human HSCs is a current topic of interest that has important implications for clinical HSC transplantation as well as basic research on HSCs. Recently, human cord blood (CB)-derived CD34− HSCs, a counterpart of murine CD34low/− KSL cells, were successfully identified using an intra-bone marrow injection (IBMI) method. This review aims to update the concept of the immunophenotype and functional characteristics of human primitive CD34− HSCs. In addition, the significance of the application of the IBMI technique in clinical CB stem cell transplantation is also discussed. Recent rapid advances in understanding the biological nature of HSCs may make it possible to fully characterize the most primitive class of human HSCs, thereby clarifying the human HSC hierarchy, in the near future.

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.