Hideo Ema

Comparison of Hematopoietic Activities of Human Bone Marrow and Umbilical Cord Blood CD34 Positive and Negative Cells

Although the hematopoietic activities of human CD34+ bone marrow (BM) and cord blood (CB) cells have been well characterized, the phenotype of nonobese‐diabetic severe combined immunodeficient (NOD/SCID) mice repopulating cells (SRCs) in CB and BM has not yet been fully examined. To address this issue, various hematopoietic activities were compared in terms of total and CD34+ CB and BM cells. Clonal culture of fluorescence‐activated cell sorter (FACS) CD34+ CB and BM cells revealed a higher incidence of colony‐forming cells with greater proliferation capacity in CB over BM CD34+ cells. CB CD34+ cells also demonstrated higher secondary plating efficiency over BM cells. In addition, we demonstrated that mice transplanted with CB mononuclear cells (MNCs) showed significantly higher levels of chimerism than those transplanted with BM MNCs. However, recipients of FACS‐sorted CD34+ CB cells showed significantly lower levels of chimerism than those that received total CB MNCs, suggesting a role of facilitating cells in the CD34− cell population. To further analyze the role of CD34− cells, the NOD/SCID repopulating ability of FACS‐sorted CB CD34−c‐kit+Lin− and CD34−c‐kit‐Lin− cells were examined. However, SRCs were not detected in those cells. Taken together, these data suggest that CB is a better source of hematopoietic stem cells and that there are cells in the CD34− fraction that facilitate repopulation of hematopoiesis in the NOD/SCID environment.

Lentiviral vector-mediated transduction of murine CD34- hematopoietic stem cells

OBJECTIVE Efficient gene transfer into murine hematopoietic stem cells (HSCs) provides a powerful tool for exploring hematopoietic stem cell biology. In this study, we evaluated the efficiency of lentiviral vector-mediated gene transfer into murine CD34(-/low)c-Kit(+)Sca-1(+)Lin(-) (CD34(-) KSL) cells that are highly enriched for HSCs. MATERIALS AND METHODS FACS-sorted CD34(-) KSL cells were transduced with the vesicular stomatitis virus G glycoprotein-pseudotyped HIV-1-based lentiviral vector containing the green fluorescent protein (GFP) gene under the control of the cytomegalovirus promoter, and then 50 transduced cells were transplanted into lethally irradiated mice. Transduction efficiency was assessed by FACS analysis for GFP expression in peripheral blood (PB) cells. FACS-sorted GFP(+) KSL bone marrow (BM) cells from primary recipients were used for secondary transplantation, and GFP expression in PB cells of reconstituted mice was analyzed by FACS. RESULTS GFP expression was detected in PB cells of all primary recipients (n = 10) at an average of 40% (range 26-58%) when the lentiviral vector containing the woodchuck hepatitis virus posttranscriptional regulatory element was used. GFP(+) cells were found in multilineage cells in PB, BM, spleen, and thymus for at least 8 months posttransplantation. In secondary recipients, donor-derived GFP(+) KSL BM cells could reconstitute and GFP expression was detected in both myeloid and lymphoid cells in PB. CONCLUSION Our results indicate that lentiviral vectors can efficiently transduce highly enriched murine HSCs and sustain long-term expression of the transgene in the multilineage differentiated progeny in reconstituted mice.

Further Characterization of CD34‐Low/Negative Mouse Hematopoietic Stem Cells

Abstract: We have previously reported that in adult mouse bone marrow, CD34low/− c‐kit+ Sca‐1+ lineage markers negative (Lin−) (CD34−KSL) cells represent hematopoietic stem cells with long‐term marrow repopulating ability whereas CD34+ c‐kit+ Sca‐1+ Lin− (CD34+KSL) cells are progenitors with short‐term reconstitution capacity. To further characterize cells in those two populations, relative expression of various genes wereee examniend by reverse transcriptase polymerase chain reaction (RT‐PCR). In CD34−KSL Cells, none of the genes sturied was found to be expressed with the exception of GATA‐2, IL‐1Rα, IL‐2Rγ, AIC‐2B, c‐kit, EPO‐R, and c‐mpl. In contrast, expression of GATA‐1 and all cytokine receptor genes examined except IL‐2Rβ, IL‐7Rα and IL‐9Rα were found in CD34+KSL.

Quantitative assessment of the stem cell self-renewal capacity.

Abstract: Little is known about the manner in which hematopoietic stem cells (HSCs) self‐renew. To address this issue, we used a serum‐free single‐cell culture, followed by transplantation of cultured cells into lethally irradiated mice. CD34‐negative or low, c‐Kit‐positive, Sca‐1‐positive, lineage marker‐negative (CD34−KSL) cells are highly enriched for murine bone marrow HSCs. Successful long‐term reconstitution with a single CD34−KSL cell enabled us to study in vitro self‐renewal of HSC at clonal level. Using this clonal cell transplantation system, we examined the effect of various cytokines on CD34−KSL cells. Among the cytokines examined, stem cell factor (SCF) and thrombopoietin (TPO) were minimum cytokines to induce cell division of CD34−KSL cells most efficiently. Similarly, multilineage repopulating activity was detected in the cells derived from a significant portion of single cells after culture in the presence of TPO and SCF. However, SCF + IL‐3, SCF + IL‐6, or SCF + IL‐11 + FL appeared to be less effective for self‐renewal of HSCs. The activity of HSCs as indicated by repopulation unit (RU) remaining after culture with SCF and TPO was not so different from that of freshly isolated HSCs. However, there was a substantial loss of HSC number in these cultured cells. Taken together, this study provides definitive proof that one HSC can generate at least one HSC in vitro.

An All-Recombinant Protein-Based Culture System Specifically Identifies Hematopoietic Stem Cell Maintenance Factors

Summary Hematopoietic stem cells (HSCs) are considered one of the most promising therapeutic targets for the treatment of various blood disorders. However, due to difficulties in establishing stable maintenance and expansion of HSCs in vitro, their insufficient supply is a major constraint to transplantation studies. To solve these problems we have developed a fully defined, all-recombinant protein-based culture system. Through this system, we have identified hemopexin (HPX) and interleukin-1α as responsible for HSC maintenance in vitro. Subsequent molecular analysis revealed that HPX reduces intracellular reactive oxygen species levels within cultured HSCs. Furthermore, bone marrow immunostaining and 3D immunohistochemistry revealed that HPX is expressed in non-myelinating Schwann cells, known HSC niche constituents. These results highlight the utility of this fully defined all-recombinant protein-based culture system for reproducible in vitro HSC culture and its potential to contribute to the identification of factors responsible for in vitro maintenance, expansion, and differentiation of stem cell populations.

Dominant expansion of human T cells in non-obese diabetic/severe combined immunodeficiency mice implanted with human bone fragments.

OBJECTIVE To establish an in vivo animal model in which human T cells develop and function normally, a step toward developing new vaccines or chemical compounds that modulate immune functions and toward understanding T-cell immunity in humans. MATERIALS AND METHODS Human bone fragments were implanted into non-obese diabetes/severe combined immunodeficiency (NOD/SCID) mice. The presence of human blood cells in the peripheral blood of these mice was monitored periodically by immunostaining and fluorescence-activated cell sorting. RESULTS After implantation of bone fragments, dominant expansion of human T lymphocytes, rather than myeloid and B cells, was observed over a 3-month period. In some cases, the proportion of human T cells rose to 40% of the peripheral blood mononuclear cells. These T cells showed CD4/CD8 ratios similar to those observed in human peripheral blood lymphocytes and had a broad repertoire of rearranged T-cell receptor genes. Graft-versus-host reaction was not noted in any organ analyzed. To assess the suitability of NOD/SCID mice implanted with human bone fragments (hu-bone-NOD/SCID mice) as an in vivo model for HIV infection, the mice were infected with a T-lymphotropic strain of HIV-1 (NL4-3) at 7 weeks posttransplant. Serum p24 gag was detected at 2 weeks after inoculation, after which total CD4-positive cell numbers declined, as seen clinically in patients infected with HIV. CONCLUSION Although the precise mechanism is yet to be determined by which predominant expansion of human T cells occurs in hu-bone-NOD/SCID mice, such mice appear likely to serve as a useful and versatile model for studies involving human T-cell immunity.

30 – Phenotype of Mouse Hematopoietic Stem Cells

In the adult mouse bone marrow, the frequency of hematopoietic stem cells (HSCs) is estimated to be 1 in 10 4 ∼10 5 cells. It is thus necessary to isolate HSCs from the other cells for their characterization. HSCs are functionally defined by their capabilities of self-renewal and multilineage differentiation. Long-term multilineage repopulation cells only measurable by experimental transplantation are considered to represent HSCs. It has recently been shown that more than 30% of such purified cells show long-term repopulating activity. This high degree of purification has enabled one to analyze HSCs at the clonal level. Accordingly, the functional heterogeneity in a stem cell population has begun to emerge. On the other hand, transcriptional profiling of purified cells has become available. Molecular analysis for stem cell function will be a focus of attention in the coming years.

Isolation and Characterization of CD34-Low/Negative Mouse Hematopoietic Stem Cells

We have previously reported that, in adult mouse bone marrow, CD34low/− c-Kit+ Sca-1+ lineage markers negative (Lin−) (CD34−KSL) cells represent hematopoietic stem cells with long-term marrow repopulating ability whereas CD34+ c-Kit+ Sca-1+ Lin− (CD34+KSL) cells are progenitors with short-term reconstitution capacity. To characterize these two populations of cells further, relative expression of various genes was examined by RT-PCR. In CD34−KSL cells, most cytokine receptor genes were not expressed with the exception of IL2Rγ and AIC-2B. In contrast, expression of all cytokine receptor genes examined except IL-2Rα, IL-7Rα, and IL9Rα chains were found in CD34+KSL cells. Cell cycle studies revealed only 3% of CD34−KSL cells and 26% of CD34+KSL cells are dividing at a given time. Long-term BrdU administration study demonstrated, however, that majority of CD34−KSL cells contribute to hemopoiesis by dividing very slowly. Furthermore, analysis of aged mice revealed more than tenfold increase in absolute number of CD34−KSL cells. Those CD34−KSL cells in aged mice appeared to include HPP-CFC at an equivalent frequency with those in younger mice. These data support our previous notion that CD34−KSL cells are at higher rank in hematopoietic hierarchy than CD34+KSL cells. In addition, our results provide important clues for cell therapy and gene therapy targeting hematopoietic stem cells.

Identification of CD34lo/−kit+sca+lin− cells in HOECHST33342 side population (Sp) Of the mouse bone marrow cells

Abstract We have previously shown that long-term marrow repopulating activity is exclusively found in CD34 lo/− Kit + Sca + Lin − cells representing 0.004% of total bone marrow cells (Science 273: 242, 1996). On the other hand, Goodell et al. reported 1000-fold enrichment of HSC activity in Hoechst 33342-stained side population (SP) of the mouse bone marrow cells (J. Exp. Med. 183:1797, 1996). In order to investigate how these two populations overlap each other in terms of stem cell activity, adult mouse bone marrow cells were first stained with Hoechst 33342 dye then by a mouse CD34 monoclonal antibody. SP cells consisted of 0.05 ∼ 0.1% of bone marrow total mononuclear cells. FACS analysis of those cells revealed that 84.9 ± 8.5% of them were positive (CD34 + SP cells) and 15.1 ± 8.5% were negative (CD34 − SP cells) for CD34. When CD34 + and CD34 − SP cells of C57BL/6 mice were sorted and transplanted into lethally irradiated C57BL/6.Ly5.1 congeneic mice (100 sorted cells/head), long-term marrow repopulating activity was found in the CD34 negative but not in CD34 positive fraction. More recently, we have established a 6-color FACS system in which direct comparison of CD34 lo/− Kit + Sca + Lin − cells and SP cells is possible. As expected, a large overlap between CD34 lo/− Kit + Sca + Lin − cells and SP cells was found. However, there are considerable number of cells that are not CD34 lo/− Kit + Sca + Lin − cells in SP fraction. Differentiation potentials of those non-hematopoietic cells are of primary interest.