hCD34 expression in the HSC fraction in bone marrow cells of transgenic mice. (a) hCD34 genomic PAC constructs. These constructs were used to establish transgenic mouse lines. (b) Expression of hCD34 in mice harboring genomic hCD34 (PAC54A19). hCD34 was expressed almost exclusively in the Lin^−/lo fraction of bone marrow cells. Within the Lin^−/lo bone marrow cells, a majority of hCD34⁺ cells were found within the c-Kit⁺ fraction. (c) More than 98% of Lin^−/loSca-1⁺c-Kit⁺ cells expressed hCD34 irrespective of mCD34 expression in both hCD34 (PAC88L2) and hCD34 (PAC54A19) transgenic mouse lines.

Expression of the hCD34 transgene in different defined HSC populations. (a) The Lin^−/loSca-1⁺ fraction (I) in hCD34 (PAC54A19) transgenic mice contains both hCD34⁻ and hCD34⁺ cells. hCD34⁺ cells are enriched in Lin⁻Sca-1⁺c-Kit⁺ fraction (II), which contains Thy1.1^lo cells. (b) The Hoechst-exporting SP was purified, resorted, and subjected to five-color fluorescence-activated cell sorter analysis. A majority of SP cells were Lin⁻Sca-1⁺c-Kit⁺hCD34⁺. (c) Changes in expression of mCD34 and hCD34 in day 14 p.c. fetal liver and from bone marrow in 4-, 10-, and 16-week-old hCD34 transgenic mice. The percentage of mCD34⁻ cells within the Lin⁻Sca-1⁺c-Kit⁺ fractions appeared to increase with age, comparing young 4-week to older 10- and 16-week-old mice. Nonetheless, more than 98% of Lin⁻Sca-1⁺c-Kit⁺ cells were positive for hCD34 irrespective of age.

Long-term reconstituting potential of mCD34^−/lohCD34⁺ HSCs. (a) Gates for sorting and results of reanalysis of purified mCD34^−/lohCD34⁺ and mCD34⁺hCD34⁺ cells within Lin⁻Sca-1⁺c-Kit⁺ HSCs. (b) Sequential changes in percentages of donor-derived blood cells after transplantation. Thirty-five mCD34^−/lohCD34⁺ (I) or mCD34⁺hCD34⁺ (II) phenotypic HSCs purified from hCD34 (PAC54A19) C57B6-Ly5.2 transgenic mice were transplanted into lethally irradiated C56B6-Ly5.1 mice. Long-term reconstitution of all hematopoietic lineages was observed exclusively in mice transplanted with mCD34^−/lohCD34⁺ HSCs. Summarized data are shown in Table 1.

Down-regulation of the hCD34 transgene in myeloid and lymphoid differentiation pathways. (a) Mouse myeloid progenitors including Lin⁻Sca-1⁻c-Kit⁺CD34⁺FcgRII/III^lo CMPs, Lin⁻Sca-1⁻c-Kit⁺CD34⁺FcgRII/III^hi GMPs, and Lin⁻Sca-1⁻c-Kit⁺CD34⁻FcgRII/III^lo MEPs (17) purified from hCD34 (PAC88L2) C57B6 transgenic mice were reanalyzed to test expression of the hCD34 transgene. A vast majority of CMPs expressed hCD34. In contrast, ≈30% of mCD34⁻ MEPs still maintain hCD34, whereas ≈20% of mCD34⁺ GMPs down-regulated hCD34. (b) Purified CLPs and the earliest thymic precursor (TP) were positive for both mCD34 and hCD34. In preT and preB stages, both mCD34 and hCD34 were down-regulated completely. The percentages of hCD34⁻ cells were higher than those of mCD34⁻ cells in both proT and proB stages, suggesting that the down-regulation of the hCD34 transgene occurs slightly earlier than that of mCD34.

Proposed differential regulation of human and murine CD34 in steady-state hematopoiesis. In steady-state human bone marrow, we propose that a majority of long-term HSCs express hCD34. In contrast, in murine hematopoiesis, mCD34 is down-regulated in the long-term HSCs, although mCD34 may be up-regulated in some fraction of long-term HSCs because of a variety of activation signals. hCD34 expression is maintained at the CMP and CLP stages. The down-regulation of hCD34 occurs earlier as compared with mCD34 in the granulocyte/macrophage and lymphoid differentiation pathways but occurs later in the megakaryocyte/erythrocyte pathway.