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Secreted MEF factors maintain HSC cultures
factors indicated. After culture, LSK cells were permeabilized and stained using the FITC Annexin V Apoptosis Detection Kit I according to the manufacturer’s instructions (BD-Biosciences).
Statistical evaluation
For statistical evaluation of the experiments, the non-parametric Mann-Whitney U test was used (Prism, GraphPad Software). A P value <0.05 was considered statistically significant. Data are pre- sented as dot plots of columns with means ± standard deviation.
Results
Here, we describe cultures in which sorted single CD34- SLAM cells from the bone marrow of B6 or 129Ly5.1 mice were cultured either in SFM, or in serum-free MEF-CM sup- plemented with either 4GF (SCF, IL-11, NGF, and Col1) or 2GF (SCF and IL-11). The single cell cultures were moni- tored for the presence and number of cells each day for a total of 5 days (Figure 1A). These experiments showed that in SFM 4GF the number of wells with dividing cells was increased compared to those in SFM 2GF (60% vs. 42%, respectively) (Figure 1B). Although in the current experi- ments SFM 4GF yielded lower survival than previously reported,1 our results show that the extra addition of NGF and Col1 to SFM 2GF improved survival of murine CD34- SLAM cells.
To further define stromal cell factors substituting CM in HSC cultures, we realized that additional sources of CM will be needed to optimally define factors maintaining HSC self-renewal. Hence, we chose to study a robust, readily available and non-transformed source of mid-gestation embryonic stromal cells: MEF. Considering that UG26-1B6 was the only cell line consistently supporting HSC in non- contact cultures,6,7 we not only added 2GF but also explored adding 4GF to MEF-CM. In a first series of experiments, we found that CM from E11.5 or E13.5 MEF with 4GF support- ed cell cycle recruitment of single CD34- SLAM cells to a similar extent (Online Supplementary Figure S1A). Since E13.5 embryos used for support of embryonic stem cells are typ- ically isolated at E13-E14,15 consistently yield more cells than E11.5 embryos, and MEF-CM from both sources behaved similarly, we performed all following experiments with E13.5 MEF-CM. To assess the variability of different MEF-CM, we prepared MEF from seven individual embryos and found that although at the beginning of cul- ture recruitment into cell division varied among different individual MEF-CM preparations, 4 and 5 days after the start of cultures, the growth kinetics were remarkably con- sistent (Online Supplementary Figure S1B).
To further optimize cell cultures, we compared CM gen- erated from freshly isolated MEF or from previously frozen MEF. Here we found that CM from both fresh and previ- ously frozen MEF stimulated cell cycle recruitment and maintenance of proliferation to a similar extent (Online Supplementary Figure S2A). Another important issue was to consider whether CD34- SLAM cells from different mouse strains we routinely use in transplantation assays3,14,16 would differ in culture with MEF-CM. We found that CD34- SLAM cells isolated either from B6 or 129Ly5.1 mice showed indistinguishable results in terms of proliferation and the number of dividing cells (Online Supplementary Figure S2B).
When comparing single cell cultures with MEF-CM 2GF and 4GF, we found that although survival was similar in
MEF-CM with 2GF or 4GF (Figure 1C, D), MEF-CM 2GF stimulated the cell cycle more efficiently compared to MEF- CM 4GF (Figure 1E). Indeed, cultures with MEF-CM 2GF showed larger clones at days 2 and 3 of culture (Figures 1C, E), while the time to first division was similarly shortened (Figure 1F), resulting in comparable increased clone sizes (Figure 1G) for both MEF-CM conditions compared to SFM 4GF. Overall, the percentage of wells with cells, wells with dividing cells, and mean clone size were significantly increased when using MEF-CM with either 2GF or 4GF as compared with SFM 4GF (Figure 1C-E, G).
To study whether these differences in number of dividing cells were attributable to cell death, we determined apopto- sis in cultures of LSK cells under SFM 4GF, MEF-CM 2GF or MEF-CM 4GF conditions. These experiments show that after 48 h of bulk culture, LSK cells cultured in MEF-CM 2GF maintained a higher percentage of viable cells and a lower percentage of apoptotic cells compared with cells cul- tured in MEF-CM 4GF or SFM 4GF, confirming the positive effect of MEF-CM for survival (Figure 2A, B).
Flow cytometric analyses of pooled clones from single cell cultures may reveal patterns of differentiation behavior as well as retention of LSK phenotypes.16 Our experiments showed that the relative numbers of CD11b+ Gr1med and Gr1hi myeloid cells were unchanged in all three culture con- ditions (Figure 2C, D, Online Supplementary Figure S3 for iso- type controls). However, the number of LSK cells recovered after 5 days of single cell culture was consistently higher in cultures with MEF-CM 2GF (Figure 2E, 2F). In addition, cells from MEF-CM 2GF cultures showed a significantly increased number of colonies in semi-solid medium com- pared with those from MEF-CM 4GF and SFM 4GF cultures (Figure 2G). These results suggest that whereas myeloid dif- ferentiation progresses at similar rates under all culture con- ditions, MEF-CM 2GF appears to maintain the HSC pheno- type and progenitor function best in culture.
To determine whether this culture condition would also support self-renewal of murine CD34- SLAM cells with repopulating ability, we collected wells containing divided cells after 5 days of culture and transplanted pools of 20 of these clones per lethally irradiated recipient (Figure 3A). Analysis of donor engraftment in the peripheral blood after 5, 10 and 16 weeks showed increased engraftment of donor cells from cultures in MEF-CM 2GF as compared to the MEF-CM 4GF or SFM 4GF-cultured CD34- SLAM cells (Figure 3B). At the early 5-week timepoint, engraftment from MEF-CM 2GF cultures was particularly prominent, suggestive of production of short-term repopulating cells in culture, followed by a stable level of engraftment at later time points (Figure 3B). With regard to differentiation of the transplanted clones, donor HSC cultured in either MEF-CM 4GF or 2GF showed higher lymphoid engraftment through- out the 16-week observation period (Figure 3C). Furthermore, donor HSC cultured in MEF-CM 2GF showed a high level of myeloid engraftment at all time points as compared with HSC cultured in MEF-CM 4GF or SFM 4GF (Figure 3D).
As in peripheral blood, donor engraftment in the bone marrow was highest in mice repopulated with divided donor CD34- SLAM cells from cultures in MEF-CM 2GF (Figure 3E, Online Supplementary Figure S4A). More impor- tantly, these mice also showed the highest regeneration of the donor-derived CD48- LSK cells (Figure 3F). Compared to mice receiving cells from SFM 4GF cultures, both cultures under MEF-CM 4GF and 2GF conditions showed higher
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