Mesenchymal Stromal Cell Co-Culture Enhances Insulin-Like Growth Factor Binding Protein 2 - Augmented Ex Vivo Cultures of Human Cord Blood in a Viability Supporting, Contact-Dependent Process Involving the Rescue of Cells from Early Apoptosis
Authors: Chu Pat PY , Prasath Arun , Li Zhihong , Bari Sudipto , Ang Justina ML , Fan Xiubo , Zhang Chengcheng , Lodish Harvey F. , Hwang William YK 
Published: April 2020

Abstract
The use of cord blood (CB) for adult transplantation is limited by the number of hematopoietic stem (HSC) and progenitor cells (HPC) residing in the CB. Successful ex vivo expansion of HSC and HPC may overcome this limitation. An ex vivo expansion system (Zhang et. al., 2008, Blood) based on the additional of Angiopoietin-like 5 and/or Insulin-like Growth Factor Binding Protein 2 (IGFBP2) to a standard cytokine cocktail of stem cell factor, thrombopoietin and FLT3-ligand has recently been reported to expand CB HSC by ~20-fold in a SCID-repopulating cell (SRC) assay. In this study, we further investigated if co-culture of the CB cells with mesenchymal stromal cells (MSC) would enhance the ex vivo expansion of CB in IGFBP2-augmented cultures. Using thawed unselected CB cells, IGFBP2-augmented cultures were able to expand CD45(+)/CD34(+) cells 49-fold and retain in vitro colony-forming units granulocyte-macrophage (CFU-GM) colony size with a concomitant 15-fold increase in number. Under the same conditions, co-culture with MSC resulted in a further 2.9-fold enhancement of CD45(+)/CD34(+) cell expansion (144-fold expansion) and a 2.2-fold enhancement of CFU-GM expansion (33- fold expansion). The numbers reflect true expansion of cell numbers as no prior CD34/ CD133 selection was employed. We further studied the mechanism of MSC enhancement of HSC and HPC expansion. Rather than an increase in cellular proliferation rate, the MSC enhancement effect was associated with 2- to 5-fold increase in viability of a population of lymphocyte-like cells [FS(low) SS(low) CD45(+)] in CB, as determined by annexin-V/7AAD staining. Moreover, direct contact with MSC was necessary for optimal expansion and viability support as shown by use of conditioned media and transwell assays. Thawed mononucleated CB cells exhibited a high level of positivity for Annexin-V, which translated to subsequent 7AAD positivity during ex vivo expansion. MSC co-culture resulted in reversion of Annexin-V positivity within 3–6 hours, and reached a maximum on day 3 (80–90% viability with MSC versus 40–60% without, as defined by double negative staining for Annexin-V and 7AAD). Concomitantly, the loss of mitochondrial membrane potential as determined by JC-1 was also reduced by MSC co-culture. There was a reduction of Caspase 9 activity (12.7% with MSC versus 23.8% without) at day 3 and a reduction in Caspase 3 and 7 activity (5.5% with MSC versus 15.5% without) at day 7 with MSC co-culture. In further experiments, an increase of lysosomal activity in CB, as determined by acridine orange staining, was found to be reduced in MSC co-culture. The use of a non-viable MSC layer did not support CB viability and subsequent expansion. We hypothesized that cytosolic transfer of nutrient or other survival factors from MSC to CB could be a potential mechanism responsible for the rescue of thawed CB from loss of viability and subsequent enhancement in CB ex vivo expansion. Using genetically modified stromal cells expressing with the fluorescent protein containing mitochondria targeting sequence (DsRed-mito) and time-lapse (&gt;10 hours) confocal imaging, we observed potential cytosolic transfer activities between the stromal cells. In conclusion, IGFBP2-augmented cultures result in successful CB expansion, which is further enhanced by MSC co-culture in a viability supporting, contact-dependent process that could involve reversal of cells from early apoptosis possibly mediated through cytosolic transfer of nutrient or cellular material. Further in vivo experimentation is in progress to verify the effect of our combined MSC-IGFBP2 culture system on HSC expansion.