Natural killer cell precursors in the CD44neg/dim T-cell receptor population of mouse bone marrow

Natural killer (NK) cells develop from the nonadherent cell component of NK long-term bone marrow (BM) cultures (NK-LTBMC). Because these nonadherent cells are depleted of mature NK cells and T cells, but appear to enriched for NK precursors, they were used as a starting population to begin to define the NK precursors that function in NK- LTBMC. As the stromal cell component of NK-LTBMC has been shown to support interleukin (IL)-2-induced, CD44 dependent, NK cell development from nonadherent NK precursors, NK-LTBMC stroma was used in a limiting dilution assay (LDA) to quantitate the precursors. NK-LTBMC in 96-well plates were irradiated (20 Gy) to kill hematopoietic cells (including the NK precursors), seeded with limiting dilutions of the cells to be quantitated, cultured with 500 U/mL IL-2 for 13 days and assayed for development of NK activity by adding 51Cr-labeled YAC-1 cells to the wells and evaluating the release of 51Cr after 4 hours. Flow cytometric analysis, sorting, and quantitation of the nonadherent cell component of NK-LTBMC showed that NK precursors were concentrated in the CD44neg/dim subset that comprised 10% of the “lymphoid” gated cells. When the CD44neg/dim subset was sorted from BM of mice treated with 5- fluorouracil (5-FU) day before (-1FUBM), there were about 30% T cells, but no NK-1.1+ cells. When the T cells were removed by sorting and the CD44neg/dim, alphabeta, gammadelta T-cell receptorneg (TCR-) subpopulation was seeded onto irradiated stroma with IL-2, they proliferated, developed NK activity, became NK-1.1+ and CD44bright and remained alphabeta, gammadelta TCR-. The frequency of NK precursors in this population as estimated from the LDA was about 1/500.

Hepatocyte growth factor as a hematopoietic regulator

Hepatocyte growth factor (HGF) was originally isolated as a mitogen for adult hepatocytes, but this cytokine is now regarded as a multi-functional factor. In the present study, we show that the mouse liver in the middle and/or late stage of the fetal life expresses both HGF and c-met (its receptor) messages. HGF and c-met mRNA are coexpressed not only in the adherent layers of fetal liver long-term cultures (FL-LTCs) and adult bone marrow long-term cultures (BM-LTCs), but also in the stromal cell lines MS-5 and PA-6. Addition of human HGF (2 and 20 ng/mL) to the LTCs enhances (1) nonadherent cell counts (ninefold in FL-LTCs and sixfold in BM-LTCs), (2) nonadherent colony-forming unit-in culture (CFU-C) counts (eightfold in FL-LTCs and fivefold in BM-LTC), and (3) cobblestone colony counts. However, HGF slightly inhibits the proliferation of stromal cells. No direct effect of HGF on freshly isolated BM and/or FL cells is found in the CFU-C assay. However, an approximately 1.5-fold synergistic increase in CFU-C counts is noted when the BM or FL cells are cocultured with HGF in the presence of interleukin-3. These findings strongly suggest that HGF plays a crucial role as a hematopoietic regulator in the proliferation and differentiation of hematopoietic progenitors.

Purified murine hematopoietic stem cells function longer on nonirradiated W41/Wv than on +/+ irradiated stroma

Mouse bone marrow (BM) was separated into low-density, lineage- negative, wheat germ agglutinin-positive (WGA+), Rhodamine-123 bright (Rhbright) or dim (Rhdim) cells to obtain populations that were highly enriched for committed progenitors (Rhbright cells) or for more primitive stem cells (Rhdim). When 2,500 Rhbright or Rhdim cells were seeded onto 6-week-old irradiated (20 Gy) long-term BM cultures (LTBMC), the nonadherent cell production from Rhbright cells was transient and ended after 5 weeks. Production from Rhdim cells did not begin until week 3, peaked at week 5, and ended at week 8, when the irradiated stroma seemed to fail. Termination of cell production from Rhdim cells did not occur in nonirradiated LTBMC from W41/Wv mice. During peak nonadherent cell production, 25% to 30% of the cells in the nonirradiated LTBMC from W41/Wv mice had donor cell markers. Two approaches were tested to try to enhance the proportion or number of donor cells. Addition of Origen-HGF at the time of seeding Rhdim cells caused a nonspecific increase in both host and donor cell production, but a specific increase in production of donor cells was obtained by seeding the cultures at 2 weeks rather than 6 weeks. Limiting dilution of Rhdim cells gave the same frequency of wells producing cells on both irradiated +/+ and nonirradiated W41/Wv or W/Wv cultures.