MenSCs efficiently support the proliferation of CD34+CD133+ hematopoietic stem cells. Cord blood CD34+ cells were immunomagnetically isolated using CD34 beads, then cultured alone or co-cultured at a ratio of 1:4 to 1:5 in different feeder layers from BM-MSCs and MenSCs (contact conditions) or separated by a transwell (TW) system (non-contact conditions). Total number of cells, the expression of CD34+CD133+ hematopoietic stem cells (HSCs) and CD34+ hematopoietic progenitors cells (HPCs) were evaluated at different time points. (A) In vitro expansion of CD34+ hematopoietic progenitor cells. Starting from three days post-culture, a statistically significant difference in favor of HSCs co-cultured with MenSCs was detected. (B) In vitro expansion of CD34+CD133+ HSCs. The in vitro expansion of HSCs showed a 3.31-fold increase (±0.33) with respect to the initial cell numbers when they were co-cultured on the MenSCs-feeder layer. (C) In vitro expansion of total hematopoietic cell numbers. The graph represents the increase of the total cell number obtained in the co-culture on the MenSCs-feeder compared to the BM-MSCs-feeder or alone. Data represent mean ± SE (*P ≤0.05 compared to BM-MSCs; #P ≤0.05 compared to HSCs). (D) Representative FACS analysis seven days post-culture (Dot plot). Percentage of the HSCs at day 1 and day 7. (E) Representative morphology of total hematopoietic cells in co-culture with BM-MSCs and MenSCs. The MenSCs-feeder layer showed a higher capacity to increase the proliferation of HSCs compared to BM-MSCs. Original magnification x10. (F) Analysis of the effect of non-contact expansion conditions. The ex vivo expansion of CD34+ cells, CD34+CD133+ cells, and total cells decreased under the non-contact condition in comparison with the cell contact cultures. Values are expressed as mean ± SE (*P ≤0.05; ***P ≤0.001). Data shown are representative of multiple replicates. BM-MSCs, bone marrow-derived mesenchymal stem cells; MenSCs, menstrual-drived stem cells; SE, standard error.