Long-term culture-initiating cell expansion is dependent on frequent medium exchange combined with stromal and other accessory cell effects

Abstract

Despite considerable effort, the expansion of long-term culture- initiating cells (LTC-ICs) in cultures of purified hematopoietic cells has not yet been achieved. In contrast, LTC-IC expansion has been attained in cultures of bone marrow mononuclear cells (MNC) using frequent medium exchange. The use of frequent medium exchange was, therefore, examined in cultures of CD34-enriched cells. In stromal- free, CD34-enriched cell cultures, medium exchange intervals ranging from 2 days to no feeding for 14 days gave similar results. Six different growth factor combinations, reported by other groups to give optimal expansion of CD34-enriched cells, were tested in comparison with the control combination of IL-3/GM-CSF/Epo/SCF. None of the combinations resulted in improved colony-forming unit-granulocyte macrophage (CFU-GM) expansion or LTC-IC maintenance, although two were equivalent. All stromal-free cultures resulted in loss of LTC-IC to half of input. Because of the limited effect of medium exchange and growth factor variations on CD34-enriched cell cultures, the effect of preformed stroma was next examined. Preformed stroma increased cell (3- fold), CFU-GM (5-fold), and LTC-IC (3-fold) output, but only when the medium was exchanged every other day. Under these conditions, the number of LTC-IC was maintained near input level. The lack of LTC-IC expansion in CD34-enriched cell cultures prompted experiments to examine the effect of cell purification. Parallel cultures were performed at CD34+lin- cell purities of 20%, 40%, 70%, and 95%, with each well containing exactly 4,000 CD34+lin- cells in addition to the CD34- accessory cells required to give the desired percentage. Also, MNC from the same source (approximately 2% CD34+lin-) were cultured at a concentration to give 4,000 CD34+lin- cells per well. As CD34+lin- cell purity was decreased from 95% to 2%, the output of cells, CFU-GM, and LTC-IC increased by threefold to fivefold. The loss of culture performance with purification was likely due to the removal of important accessory cells, because the levels of endogenously produced leukemia inhibitory factor and IL-6 were found to decline significantly with increasing CD34+lin- cell purity. In summary, preformed stroma abrogated the decrease in cell and CFU-GM output from cultured CD34- enriched cells and led to LTC-IC maintenance. In contrast, MNC inocula resulting in a growing stromal layer during the culture led to LTC-IC expansion (3.2-fold).(ABSTRACT TRUNCATED AT 400 WORDS)

Importance of Parenchymal: Stromal Cell Ratio for the Ex Vivo Reconstitution of Human Hematopoiesis

“…Culture output from CD34-enriched cells was significantly increased by the presence of IPFS, consistent with previous reports [20,28], and the magnitude of this stromal-dependency of CD34-enriched cells within a given experiment has been shown to be dependent upon characteristics of the CD34-enriched cell donor, but not the IPFS cell donor [28]. The present data further show that the stromaldependency index ([SDI] defined as the ratio of culture output with IPFS:culture output without IPFS) was dependent upon the CD34-enriched cell inoculum density as well ( Fig.…”

“…Long-term bone marrow culture (LTBMC) medium was prepared by supplementing Iscove's modified Dulbecco's medium (IMDM) with 10% horse serum, 10% fetal bovine serum (FBS), 4 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin (all from GIBCO; Grand Island, NY), and 5 µM hydrocortisone (Sigma; St. Louis, MO). Growth medium was prepared by supplementing LTBMC medium with a previously optimized mixture [20] In some experiments, the growth medium was further supplemented with conditioned medium (CM) taken from cultures of IPFS cells. CM was removed on day 7 and concentrated 10-fold in a Centriprep-10 ™ device (10 kD cut-off membrane; Amicon; Beverly, MA).…”

“…After inoculation, 50% of the culture volume was replaced on days 4, 7, 9, 10, and 11. Cells were harvested after 12 days of culture using trypsinization as previously described [20], and were then resuspended in LTBMC medium for counting and assays.…”

“…Cells were plated at 3,000 to 20,000 per ml, depending upon the expected clonogenicity. Cultures were maintained for 14 days and were then scored as previously described [20].…”

“…Enrichment of the primitive hematopoietic parenchymal elements, containing the self-renewing stem cell population, can be performed using cell selection technologies based upon expression of the CD34 surface marker [19]. However, it is known that stromal elements play an important role in determining the fate of developing hematopoietic cells [20,21], and it has been proposed that specific stem cell supportive niches exist within BM stroma [22]. These known clinical and scientific aspects of hematopoiesis make it a good model system for investigation of the relative roles of parenchyma and stroma in ex vivo human tissue reconstitution.…”