Expansion of Hematopoietic Stem Cells (HSC) from Cord-Blood (CB) Derived Mononuclear Cells (MNC) in Cytokine-Free Environment Using Mesenchymal Cells Spatial Co-Culture System.

In situ, HSCs are intimately associated with discrete spatially organized niches within the bone marrow, which are part of the “hematopoietic inductive microenvironment” (HIM). The HIM provides a range of molecular signals that collectively control HSC differentiation and self-renewal. This process is mediated via cell-cell and cell-ECM molecular contacts or through specific factors synthesized and secreted by mesenchymal stromal cells (MSC).

Current strategies aimed at ex vivo expansion of transplantable HSC have so far been met with limited success. Most attempts to expand HSC ex-vivo are based on using monolayers of MSC as a supportive tier or growth media supplemented with blend of cytokines. failure to support long-term maintenance and expansion of human HSC on MSC monolayers could be associated with inadequate physical architecture of the culture systems which does not reflect the natural 3-D growth conditions present within the BM-HIM. The other method based on supporting the Hematopoietic cells proliferation by a blend of cytokines, is proven routine but post transplantation marrow replenishment is currently unsatisfactory and chromosomal epigenetic modifications may be introduced into progeny cells.

Using the PluriX™ bioreactor, we had previously demonstrated that spatial co-cultures of MSC and HSCs provide an efficient system for the expansion of HSCs from CB CD34+ selected cells in cytokine-free environment.

Over the past years, attempts to expand HSCs mostly employed CD34+ selected cells. However, these cells may not represent the earliest HSCs and the immunomagnetic selection protocols are costly, time consuming and associated with great loss of source cells. A theoretical approach to overcome both hurdles is by using non-selected MNC as the founding population of HSC. Following our ability to expand HSCs from CD34+ selected cells, we now demonstrate the capacity of the 3-D HSC MSCs co-culture system within the PluriX™ bioreactor system to expand HSCs using MNC as HSCs source.

Primary Human marrow-derived MSCs were grown on 3-D carriers within the PluriX™bioreactor system. When the MSCs cultures reached high density (3*10⁶–8*10⁶ cells/ml) CD34+ selected cells or MNC were plated onto them. Within 7–14 days, the population of CD34+ and CD34+CD38− cell were expanded irrespective of the HSCs founding source. However, during this period of time the absolute expansion magnitude was greater when MNC rather than CD34+ selected cells were used to drive the process. The enrichment rate of CD34+cell from MNC and CD34+ selected cells was 20–100 vs. 5–20 fold, respectively. Under same conditions, the enrichment of the earlier CD34+CD38− cells was 40–50 vs. up to 10 fold for the same cell populations. In-conclusion, the spatial co-cultures of MSC and HSC within the PluriX™ bioreactor have been shown to form a potent HSCs expansion system in non-supplemented cytokine environment. The capacity of this practice is improved when MNCs, instead of CD34+ selected cells, serve as the originating population for HSCs. It is conceivable that the efficiency of this system is based upon selective adherence between HSCs and the hematopoietic niches within the MSCs 3-D cultures. As such, upscale of the current system could become attractive method for HSCs expansion.