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S. Romero Marquez et al.
proliferation rate. However, compared to the serum-free cultures, the MEF-CM shortened the time to first division. As such, the proliferation process as a whole was affected in that larger clones were detected after 5 days. The processes of survival, cell cycle (recruitment), and self- renewal are not only independently regulated in murine HSC,1 but also in human HSC.17 Thus, similarly to the UG26-1B6-derived CM, MEF-CM can be used to define stromal factors beneficial for survival and self-renewal of murine HSC, and possibly also human HSC.
In our previous study, we showed that some of those fac-
A
BCD
EFG
Figure 3. Self-renewal of repopulating hematopoietic stem cells in single cell cultures. (A) Experimental design: CD34- SLAM cells (positive for both CD45.1 and CD45.2) were sorted as single cells into 96-round-bottomed plates prefilled with SFM 4GF, MEF-CM 4GF, or MEF-CM 2GF as specified in Figure 1. To determine self- renewal of repopulating cells in cultures of single cells, 20 wells in which at least one cell division had occurred (cell clones) were harvested and transplanted into lethally irradiated (CD45.2) primary recipients together with competitor cells (CD45.2) as described in the Methods section. (B) Donor cell engraftment in peripheral blood, 5, 10, and 16 weeks after transplantation of divided CD34- SLAM cells previously cultured under the specified conditions. (C) Percentages of the lymphoid (CD3e+ and CD45R (B220)+ cells) and (D) myeloid (CD11b+ Gr1+) engraftment in peripheral blood, 5, 10, and 16 weeks after transplantation of growing clones from 5-day cultures as fractions of the total (donor + recipient = 100%) CD45+ lymphoid and myeloid compartments, respectively. (E) Percent donor engraftment in CD45+ cells of the bone marrow after transplantation of divided cells from single cell cultures. (F) Representative flow cytometric plots of donor and recipient cells in the Lineage- cell fraction from the bone marrow (upper row) and the donor-derived Lineage- fraction (lower row), showing myeloid progenitors (SCA1- KIT+) cells and LSK cells (Lineage- SCA1+ KIT+ cells; lower row), 16 weeks after transplantation. (G) Absolute number of HSC-enriched CD34- CD48- LSK donor cells from the bone marrow of mice transplanted with 20 clones each from single cell cultures. For gating of CD34- CD48- LSK cells from the LSK cells shown in the previous panel (F), see Online Supplementary Figure S4C. The absolute number of cells was calculated by multiplying relative numbers of Lineage- and LSK cells by the total number of donor cells in the bone marrow (Online Supplementary Figure S4A) The results shown here represent three independent experiments with a total of 11 recipients transplanted with divided cells from cultures in SFM 4GF, 13 recipients transplanted with divided cells from cultures in MEF-CM 4GF, and 8 recipients with divided cells from cul- tures in MEF-CM 2GF. As in Figure 1, black dots represent results for SFM 4GF, blue dots represent results for MEF-CM 4GF and red dots represent results for MEF- CM 2GF. *P<0.05 using the non-parametric Mann Whitney U-test for comparisons between SFM 4GF and either of the two MEF-CM conditions. LT-HSC: long-term hematopoietic stem cells; CM: conditioned medium; 2GF: two growth factors (i.e., stem cell factor [SCF] and interleukin-11 [IL-11]); 4GF: four growth factors (i.e., SCF, IL-11, nerve growth factor [NGF] and collagen 1 [Col1]); SFM: serum-free medium; FACS: fluorescence activated cell sorting; PB: peripheral blood; BM: bone marrow; LSK: Lineage- SCA1+ KIT+.
tors, in particular NGF and Col1, may substitute for the UG26-1B6-CM.1 Indeed, in the present study, culture of CD34- SLAM cells in SFM 4GF improved survival over cul- tures in SFM 2GF. Importantly, the MEF-CM 2GF condition promoted both survival and cell cycle recruitment of single CD34- SLAM cells compared to SFM 4GF. It is also note- worthy that cultures in MEF-CM 2GF produced significant- ly more repopulating cells and maintained cells repopulat- ing hematopoiesis at significantly higher levels throughout the engraftment period. The addition of extra NGF and Col1 to MEF-CM 2GF cultures did not additionally improve
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haematologica | 2021; 106(10)