Biological characteristics of rabbit bone marrow mesenchymal stem cells and their response to different growth factors / 中国实验血液学杂志

This study was aimed to analyze the biological characteristics of rabbit bone marrow mesenchymal stem cells (rBM-MSCs) and their response to different growth factors. Rabbit BM-MSCs were separated from bone marrow mononuclear cells by using adherent cultivation. Biological characteristics were investigated by optical and electron microscopy. Immunophenotype of rBM-MSCs was measured by flow cytometry. The expression of collagen was detected by RT-PCR. Differentiation potential was identified by specific staining and RT-PCR. The response of rBM-MSCs to IL-1, 3, 8 and HGF with different concentrations were tested by MTT. The results showed that the rBM-MSCs gave rise to a population of adherent cells characterized by the presence of a predominant cell type with a typical fibroblast-like morphology and could be cultured for over 15 passages. CD44 was highly expressed on F5 rBM-MSCs (32%) and CD45 was lowly expressed (4.7%). Type I collagen was highly expressed, while type II collagen was lowly expressed and type X collagen was not detected on rBM-MSCs using RT-PCR method. In various conditions inducting differentiation, rBM-MSCs could differentiate into the osteoblast, chondrocyte, adipocyte and neuron-like cells. The rBM-MSCs were sensitive to IL-3, even low concentration (10 ng/ml) of IL-3 could promote the proliferation of rBM-MSCs effectively (>32%, P < 0.01), whereas high concentration IL-3 inhibited it significantly. It is concluded that rabbit BM-MSCs were successfully isolated and culture-expanded. The biological characteristics of rabbit BM-MSCs are similar to those of human and rhesus BM-MSCs. IL-3 with low concentration can promote the proliferation of rBM-MSCs effectively, but high concentration of IL-3 can inhibit their proliferation.

Double chimerism in recipient by transplantation of two allogeneic MHC-mismatched mouse fetal blood units / 中国实验血液学杂志

We have constituted a mouse model for fetal blood transplantation (FBT) to cross over major histocompatibility complex (MHC) without causing serious GVHD. It seems that full matching at the MHC appears not necessary for FBT, while the nucleated cell dose is critical. Two fetal blood units were combined from different donors to increase the stem/progenitor cell dose so as to explore the possibility of MHC-mismatched allogeneic transplantation. 26 out of 40 mice in mixed FBT group survived in the observation period of 60 days after transplantation without obvious GVHD. Double chimerism was demonstrated by PCR and flow cytometric analysis; and skin transplantation test proved the induction of donor specific immune tolerance. Our data suggest that two MHC-mismatched allogeneic donor fetal blood units could simultaneously engraft and reconstitute immune and hematopoietic system in a mouse model. The result may be beneficial for the expansion of cord blood application and enables more patients to share the advantages of cord blood transplantation.

Operating Procedure of Collection, Processing and Preservation of 3000 Units Umbilical Cord Blood in Shangdong Cord Blood Bank / 中国实验血液学杂志

The experience with the umbilical cord blood (UCB) stem cells for unrelated transplantation from our 3 000 UCB storage was described. UCB, collected from closed blood bags, were mixed with hydroxyethyl starch for nucleated cell (NC) enrichment. After finishing CD34 analysis, culture of hematopoietic progenitors (CFU-GM and CFU-GEMM) assays, microbial culture, HLA Class I (A, B) serology and class II (DR) low resolution SSP typing, cord blood units are stored in the liquid nitrogen for clinical applicatoin. Cord blood contained an average of nuclear cell (NC) (1.2 +/- 0.6) x 10(9), CD34(+) cells (3.0 +/- 3.7) x 10(6), CFU-GM (1.1 +/- 0.7) x 10(6) and CFU-GEMM (1.1 +/- 1.2) x 10(6) for storage and the recovery rates were 91%, 88%, 85% and 82%, respectively. The recovery rates for red blood cell and Hb were (39 +/- 9)% and (40 +/- 8)%, respectively. The storage volume was (35.1 +/- 7.1) ml in a 50 ml storage bags. The mean time from collection to processing of 15 hours (range 4 - 24 hours) had no influence on cell viability. The cell viability before processing is more than 95% and 92% after UCB thawing. The recovery rates of NC, CD34(+) cells and CFU-GM post-thawing were 96%, 90% and 91%, respectively. There were no HIV antibody (HIVAb) positive in all of UCB units. For an incidence of processed samples, infection with syphilis, HBsAg, HBcAb, HCVAb, CMV, bacterial contamination and abnormal hemoglobin were 0.1%, 0.8%, 3.2%, 0.2%, 87.1%, 1.2% and 0.1%, respectively. More than 3 HLA loci matched can be found for random patients in our cord blood bank and 6 HLA loci matched have 5%. For transplantation with nucleated cell counts of > 2.7 x 10(7) cells/kg, our cord blood bank will be able to provide all of the umbilical cord blood stem cell samples for children and 50% of units can be used for some of adult recipients transplantation in the country. It is concluded that: (1) The large cord blood banking for 20 000 UCB storage is feasible in China. (2) Our system of whole procedure and methods is functionable for supplying qualified cord blood units in transplantation. (3) The volume for collection is critical to the yield of CD34(+) cells or hematopoietic progenitor cells, however cord blood NC is also important and proportional with CD34(+) cells. Only the units containing more than 8 x 10(8) cells and more than 60 ml of cord blood can be in the procession for storage.