Effect Of SCF On Ex Vivo Expansion And In Vivo Reconstitution Of HSCs And Its Mechanism

Hematopoietic stem cells (HSCs) derived from cord blood (CB) have several advantages such as larger hematopoietic colonies and superior ex vivo expansion capability, reduced incidence and severity of acute and chronic graft-versus-host disease, easy to collect and no harm to donors. Thus, transplantation of CB-HSCs holds great promise for clinical translation, which provided a alternative therapeutical strategy for treating various diseases. However, the very low frequency of HSCs in single CB remains a big barrier to their wider clinical applications, since both successful engraftment and fast blood reconstitution largely rely on the number of infused HSCs. To address such a challenging issue, ex vivo expansion of HSCs represents the most promising strategy, which focuses on simulating the microenvironment HSCs reside in vivo which also called niche. Stem cell factor (SCF) is proven to be an essential factor in niche so that it is widely used in HSCs ex vivo expansion. However there is none definite scheme of how to use it in HSCs ex vivo expansion, which results in the great differences of the quality and quantity of expanded HSCs and difficulties of standarizing the HSCs ex vivo expansion process. Therefore, the effect of SCF doses on CB-HSCs ex vivo expansion characteristic will be analyzed in this study. Also, the kinetic parameters will then be calculated including the specific growth rate and the SCF consumption rate to establish the relationship of SCF dose and cell expansion. Based on the analysis of kinetics, the SCF feeding scheme will be optimized. Meanwhile, the capacities of engraftment and multilineage reconstitution in vivo of epanded HSCs will be tested in NOD/SCID mouse model. Furthermore, the effect of SCF feeding regimen on gene expression of key molecules in signaling pathways induced by SCF will be studied using gene arrays, so as to understand the mechanism of SCF regulating the ex vivo expansion and in vivo engraftment of HSCs and lay an foundation for optimization of HSCs ex vivo expansion process.Firstly, four different doses of SCF including 0,5,50 and 500 ng/ml were chosen to stimulate ex vivo expansion of CB-derived CD34+ cells, and the expansion folds of total cells, the percentages of CD34+ cells and CD34+CD38+ cells and the lineage compositions of expanded cells were analyzed. It was found that the expansion folds of total cells depended on SCF doses when it ranged from 0 to 50 ng/ml, which means higher SCF doses would result in more cell expansion folds. Nevertheless there was a threshold of SCF dose beyond which could not improve cell expansion. On the other hand, higher SCF doses resulted in the faster decrease of percentages of CD34+ cells and CD34+CD38- cells, which means more SCF would promote the differentiation of HSCs. Therefore,50 ng/ml was proven optimal to expand CD34+ cells in this study, considering both the expansion of total cells and maintaining the percentages of HSCs.Further, the cell growth and the consumption of SCF were calculated to investigate the relationship between SCF dose and cell expansion. It was found that higher doses of SCF could promote cell expansion and get higher specific growth rate in the initial stage (0～4 d) during the culture process. Also, cell expansion was prolonged with higher doses of SCF. Besides, there was a positive correlation between the specific growth rate and the specific consumption rate of SCF. High specific growth rate was achieved when the specific consumption rate of SCF was at least 0.15 pg/(cellxday). These results indicated that the initial stage (0-4 d) was the key time point to stimulate the expansion of cells, and the dose of 50 ng/ml was suitable to control the SCF consumption rate of 0.15 pg/(cellxday).On the basis of above, it was hypothesized that the feeding timing of SCF was critical for HSCs ex vivo expansion so that the effect of SCF feeding regimen on HSCs expansion characteristic was explored. It was found that a novel SCF feeding regimen was effective in expanding HSCs, wherein SCF was added just into the initial medium and no SCF was supplemented during the following culture process. This approach increased the cell yield from (12.2±5.2)×104 cells/ng SCF to (44.2±24.5)×104 cells/ng SCF, which also cut down the cost of HSCs ex vivo expansion and ease the operation. Further the capacities of engraftment and mulitilineage reconstitution in vivo of expanded CD34+ cells were analyzed in NOD/SCID mice. The results showed that CD34+ cells expanded by the novel SCF feeding regimen could engraft and reconstitute in vivo successfully, but the engraftment level and colony forming ability of engrafted cells were lower than that of cells expanded with multi-feeding of SCF during all the culture process, suggesting the important role of SCF in maintaining the physiological function of HSCs.To further realize the effect of SCF feeding regimen on biological characteristics of HSCs and its regulative mechanism, gene arrays of CD34+ cells expanded by the two SCF feeding regimens, including just adding SCF into initial medium and multi-feeding of SCF at regular intervals during the culture process, were applied to explore the gene expression of key molecules in PI3K-AKT and JAK-STAT signaling pathways induced by SCF. It was found that 68 and 64 genes expressed differently in CD34+ cells expanded with SCF adding into initial medium and with multi-feeding of SCF at regular intervals compared with freshly isolated CD34+ cells, respectively. Then the relationship between the differently expressed genes and the regulation of cell expansion and movement was predicted by Ingenuity Pathway Analysis (IPA) software. The result showed that HSCs proliferation was regulated by SCF through PI3K-AKT and JAK-STAT pathways. BTK in PI3K-AKT pathway as well as CSF1R and IL4R in JAK-STAT pathway could activate HSCs proliferation, while NFKBIA in PI3K-AKT pathway as well as MPL and IFNG in JAK-STAT pathway could inhibit HSCs proliferation. These differently expressed genes led to activation of HSCs proliferation. The respectively expression difference of these genes in CD34+ cells expanded with different SCF feeding regimens compared with freshly isolated CD34+ cells was close, indicating the effects on gene expression related to HSCs proliferation was similar of different SCF feeding regimens. Meanwhile,23 genes which were expressed differently in expanded CD34+ cells compared with freshly isolated CD34+ cells in JAK-STAT pathway were related to regulation of cell movement, in which 14 genes could activate cell movement while 4 genes inhibit it. The differently expression in there genes of CD34+ cells expanded with two SCF feeding regimens both inhibit the cell movement ability, including migration, chemotaxis and homing. Moreover, five genes(NOS2, TYK2, CCND1, GATA3 and CXCL9) were expressed differently in CD34+ cells expanded with adding SCF into initial medium compared with CD34+ cells expanded with multi-feeding of SCF at regular intervals. Among these five genes, TYK2, CXCL9 and CCND1 could activate cell migration. TYK2 was up-regulated and CXCL9 as well as CCND1 were down-regulated in CD34+ cells expanded with adding SCF into initial medium, which result in its abililies of cell migration and chematoxis in CD34+ cells were inferior to those in CD34+ cells expanded with multi-feeding of SCF.Through this study, the effect of doses and feeding scheme of SCF on ex vivo expansion of HSCs and in vivo reconstituion was realized, while the relationship was understood between gene expression of key molecular in signaling pathway induced by SCF and cell proliferation as well as cell movement. The results laid foundation of how the SCF feeding regimen in ex vivo expansion system regualte biological characteristics of HSCs, which was important to establish a standard and large scale HSCs ex vivo expansion technology and expand their therapeutic applications.