ABSTRACT
Hematopoietic stem cells [HSCs] transplantation using umbilical cord blood [UCB] has improved during the last decade. Because of cell limitations, several studies focused on the ex vivo expansion of HSCs. Numerous investigations were performed to introduce the best cytokine cocktails for HSC expansion The majority used the Fms-related tyrosine kinase 3 ligand [FLT3-L] as a critical component. According to FLT3-L biology, in this study we have investigated the hypothesis that FLT3-L only effectively induces HSCs expansion in the presence of a mesenchymal stem cell [MSC] feeder. In this experimental study, HSCs and MSCs were isolated from UCB and placenta, respectively. HSCs were cultured in different culture conditions in the presence and absence of MSC feeder and cytokines. After ten days of culture, total nucleated cell count [TNC], cluster of differentiation 34[+] [CD34[+]] cell count, colony forming unit assay [CFU], long-term culture initiating cell [LTC-IC], homeobox protein B4 [HoxB4] mRNA and surface CD49d expression were evaluated. The fold increase for some culture conditions was compared by the t test. HSCs expanded in the presence of cytokines and MSCs feeder. The rate of expansion in the co-culture condition was two-fold more than culture with cytokines [P<0.05]. FLT3-L could expand HSCs in the co-culture condition at a level of 20-fold equal to the presence of stem cell factor [SCF], thrombopoietin [TPO] and FLT3-L without feeder cells. The number of extracted colonies from LTC-IC and CD49d expression compared with a cytokine cocktail condition meaningfully increased [P<0.05]. FLT3-L co-culture with MSCs can induce high yield expansion of HSCs and be a substitute for the universal cocktail of SCF, TPO and FLT3-L in feeder-free culture
ABSTRACT
Despite of many benefits, umbilical cord blood [UCB] hematopoietic stem cell [HSC] transplantation is associated with low number of stem cells and slow engraftment; in particular of platelets. So, expanded HSCs and co-transfusion of megakaryocyte [MK] progenitor cells can shorten this period. In this study, we evaluated the cytokine conditions for maximum expansion and MK differentiation of CD133[+] HSCs. In this experimental study, The CD133[+] cells were separated from three cord blood samples by magnetic activated cell sorting [MACS] method, expanded in different cytokine combinations for a week and differentiated in thrombopoietin [TPO] for the second week. Differentiation was followed by the flow cytometry detection of CD41 and CD61 surface markers. Colony forming unit [CFU] assay and DNA analysis were done for colonogenic capacity and ploidy assay. CD133[+] cells showed maximum expansion in the stem span medium with stem cell factor [SCF] + FMS-like tyrosine kinase 3-ligand [Flt3-L] + TPO but the maximum differentiation was seen when CD133[+] cells were expanded in stem span medium with SCF + Interleukin 3 [IL-3] + TPO for the first and in TPO for the second week. Colony Forming Unit-MK [CFU-MK] was formed in three sizes of colonies in the mega-cult medium. In the DNA analysis; 25.2 +/- 6.7% of the cells had more than 2n DNA mass. Distinct differences in the MK progenitor cell count were observed when the cells were cultured in stem span medium with TPO, SCF, IL-3 and then the TPO in the second week. Such strategy could be applied for optimization of CD133[+] cells expansion followed by MK differentiation
ABSTRACT
Cell vital function has correlation with mechanical loadings that cell experiences. Here, effects of in-vitro combined cyclic-static stretch on proliferation of human mesenchymal stem cell [HMSC] were evaluated. HMSCs were cultured on gelatin coated elastic membranes, and exposed to stretch loading. Four different regimes of cyclic, static, combined cyclic-static, and cyclic with a period of unloading were exerted on the elastic membrane. Duration of cyclic loading and static loading was 5 and 12 hours respectively. The results illustrate that 10% cyclic stretch causes cell alignment but there were no significant proliferation differences between control and test group. Combined cyclic-static stretch reduced proliferation significantly while cyclic stretch with an unloading period increased cell proliferation significantly. At last, static stretch did not affect cell proliferation significantly. Cell stretching regimes and post-loading duration are effective factors on cell proliferation