Mature Accessory Cells Influence Long‐Term Growth of Human Hematopoietic Progenitors on a Murine Stromal Cell Feeder Layer

Abstract

A recently described long-term culture system for early human progenitor cells was established with the murine preadipocyte stromal line FBMD-1 grown in 96-well plates; cobblestone areas formed by inoculated hematopoietic cells are determined in a limiting dilution setting after five weeks' culture. To compare the capacity of cobblestone-area-forming cell (CAFC) formation by bone marrow and leukapheresis products in this system, mononuclear cells (MNC) of both origins were cultured. As related to CD34 + cell content, CAFC yields after five weeks' culture were in the same range in bone marrow and leukapheresis stemming from patients with efficient mobilization of hematopoietic cells. In purified CD34 + cell fractions, the CAFC yield per inoculated cell number was considerably higher than in MNC; however, if the CAFC number was related to the inoculated CD34 + cell number in MNC and after purification, the yield was four to eight times decreased in purified fractions. Addition of the mature cells brought the CAFC yield back up to the numbers obtained in the unseparated MNC fraction. By contrast, slightly more advanced progenitors per CAFC were found in cultures of purified hematopoietic cells from both origins than in whole MNC. The results suggest that mature human accessory cells give noticeable support to recruitment of early progenitors on this feeder but lead to lower yield of GM progenitors.

Cell and gene therapy using mesenchymal stem cells (MSCs)

“…Previous studies have shown that preadipocytes have a higher ability to support hematopoiesis than other kinds of stromal cell components in vitro [12,13]. Our results of gene expression profile revealed up-regulation of critical cytokines for hematopoiesis such as SCF and SDF-1 in preadipocyte A54 cells.…”

Evidence for a qualitative hierarchy within the Hoechst-33342 ‘side population’ (SP) of murine bone marrow cells

“…Evidence for such accessory cells, or rare stromal elements, has been reported elsewhere. [30][31][32][33] Interestingly, Uchida et al 29 also report a disparity between calculated long-term culture-initiating cell (LTC-IC) frequencies and estimates of primitive hematopoietic progenitor cell numbers in isolated SP subsets. One possible interpretation of this observation may also be the exclusion of rare accessory cells required for optimal performance of the LTC-IC assay by the SP selection process.…”

Human Accessory Cells Have a Humoral Bystander Effect on CAFC Growing on Murine Feeder

“…As we reported earlier, growth of CAFC was impeded if human CD34 1 cells from BM and LP were enriched and cultured on FBMD-1 feeder cells complemented with IL-3 and G-CSF. This effect was reversed after adding the mature cell fraction (16), which excludes loss of early progenitors during purification and demonstrates a supporting effect of human accessory cells. It is of note that the number of CAFC formed is different for both conditions right from the beginning, and the difference is not enhanced in later stages after the fourth week.…”

“…However, growth efficiency per inoculated CD34 1 cell decreased by about one order of magnitude if enriched CD34 1 cell fractions were inoculated instead of MNC stemming from the same harvest. Optimal growth could be restored if the mature cell fraction was added back (16). These findings suggested that concomitant mature human cells improve growth conditions for early human progenitors as CAFC on murine stromal cells.…”

“…Enriched CD34 1 cell fractions were obtained by an immunomagnetic cell sorting system as described (MINI-MACS, Miltenyi) (16,21): 100 ml of CD34 monoclonal antibody (mAb QBEND-10) was added per 1 3 10 8 MNC, suspended in 200 ml of PBS-EDTA containing 0.5% bovine serum albumin (BSA), and incubated for 15 min at 4°C. Cells were washed and resuspended in 300 ml of PBS-EDTA-BSA, and 100 ml of paramagnetic beads coupled to anti-mouse mAb were added.…”