China ; Cord Blood Stem Cell Transplantation ; Fetal Blood ; cytology ; Hematopoietic Stem Cell Transplantation ; Humans

This study was aimed to explore the effect of different cryopreservation time on recovery rate of cord blood stem cells, and analyze the influence of cord blood cells after thawing on the engraftment speed of cord blood cells in patients. 20 cord blood units were stored at -196°C for 1 - 10 years. The cell viability, content of total nucleated cell (TNC), CD34(+) cells and the colony forming units of granulocyte/macrophage (CFU-GM) were assessed after thawing, the impact of cell recovery on engraftment speed in patients was analyzed. The results showed that as compared with data provided by Umbilical Cord Blood Bark, the different cryopreservation time had no effect on yield of cord blood stem cells after thawing. The cell viability was (92.75 ± 2.55)% after thawing, the yields of TNC, CD34(+) cells and CFU-GM were 89.9%, 84.8% and 84.3%, compared with that of pre-freezing, their differences were statistically significant (P = 0.000), however, loss of cells had no effect on the time of neutrophils and platelets engraftment. The TNC and CD34(+)cell count after thawing correlated closely with that of pre-freezing (r = 0.954 and r = 0.931, P = 0.000), but CFU-GM content poorly correlated with that (r = 0.285, P = 0.223). It is concluded that cryopreservation and thawing process can damage the cord blood stem cells, leading to cell loss, but not affect transplant results.
Cell Count ; Cell Survival ; Cord Blood Stem Cell Transplantation ; methods ; Cryopreservation ; methods ; Fetal Blood ; cytology ; Humans

This study was purposed to investigate the accelerating effect of newborn mouse cord blood transplantation combined with hematopoietic progenitor cells of bone marrow (BM) on the early hematopoietic reconstitution after transplantation, and the long-term chimerism of cord blood-derived cells, so as to develop a combined transplantation method for accelerating the early hematopoietic reconstitution. The lin(-)sca-1(-)c-kit(+) (c-kit(+)) cells and lin(-)sca-1(+) (sca-1(+)) cells in the bone marrow of BDF1 mice were isolated by MACS method. Biological characteristics in vitro of isolated fractions were observed and compared by semisolid colony culture combined with Giemsa staining. After transplantation of cord blood (CB) alone, or together with graded numbers of either c-kit(+) or sca-1(+) cells isolated from BDF1 mice (CD45.1) bone marrow into lethally irradiated CD45.2 congenic BDF1 mice, numbers of WBC and platelet were measured within 22 days of post-transplantation. The proportion of chimerism on granulocyte, T and B cell was dynamically measured by flow cytometry within 60 weeks of post-transplantation. The results showed that the number of colony from BM c-kit(+) cells cultured in semi-solid agar medium was significantly smaller than that from BM sca-1(+) population, which showed low proliferative potential in vitro and morphological characteristics of medium- or large-sized blast-like cells. The co transplantation of CB and BM c-kit(+) cells or sca-1(+) cells at the dosages of 1 × 10(4) or 2.5 × 10(4) or 5 × 10(4) to recipient mice leads to the quantity of WBC and platelets increased to 1 × 10(9)/L and 1 × 10(12)/L at day 12, whereas the injection of CB alone resulted at day 17. When mice were transplanted with CB together with BM c-kit(+)cells, and the CB-donor type cells in the peripheral blood increased progressively, while congenic donor BM-derived stem cells decreased gradually. After cotransplantation with CB and BM c-kit(+) cells for 60 weeks, a frequency of complete chimerism in CB-derived cells was continually maintained in granulates (96.68 ± 2.68)% and B lymphocytes (92.55 ± 3.04)%, while T lymphocytes (67.96 ± 7.91)% were dominantly derived from CB. On the other hand, congenic bone marrow or residual-derived cells were the dominant population, and the ratio of CB-derived cells in the peripheral blood was less than 10% (6.19 ± 7.62)% after cotransplantation with CB and sca-1(+)cells for 60 weeks. It is concluded that the cotransplantation of CB and BM congenic c-kit(+) cells is able to accelerate early hematopoietic reconstitution of recipient mice due to congenic marrow cells. Complete or main chimerism of cord blood is formed in long-term multilineage reconstitution of granulocytes, B cells and T lymphocytes.
Animals ; Bone Marrow Transplantation ; immunology ; Fetal Blood ; cytology ; transplantation ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells ; cytology ; immunology ; Mice ; Mice, Inbred C57BL ; Proto-Oncogene Proteins c-kit ; immunology

The hematopoietic repopulating ability of fresh and cultured CD34+ cells and CD34- cells derived from cord blood were compared by nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse model. Fresh CD34+ cells and CD34- cells were isolated from fresh cord blood. Cultured CD34+ cells and CD34- cells were separated from cultured mononuclear cells (MNC). We transplanted these cells into sublethally irradiated NOD/SCID mice via the tail vein and sacrificed surviving mice after 6 weeks. The peripheral blood, spleen and bone marrow from each mouse were harvested for flow cytometry, colony-forming cells and human Alu sequences analyses. The proportions of CD45+ cells and human multilineage hematopoietic cells in NOD/SCID mice received CD34+ cells were close to that in the mice received both CD34+ cells and CD34- cells, while it was significantly higher than that in the mice received CD34- cells. Six weeks after transplantation, all the mice injected with cultured CD34- cells dead. The survival rate of mice injected with cultured CD34+ cells was 66.7%. All of the mice injected with both cultured CD34- and CD34+ cells survived. Moreover, CD45+ cells could be detected in all surviving mice, and human CD34, CD3, CD19, CD33 and CD71 antigen also could be detected on these CD45+ cells. The results showed that both fresh and cultured CD34+ cells had the capability of engraftment and hematopoiesis reconstitution, but CD34- cells hadn't the ability. However, CD34- cells had assistant effect on the hematopoietic repopulating ability of CD34+ cells.
Animals ; Cells, Cultured ; Cord Blood Stem Cell Transplantation ; Embryonic Stem Cells ; cytology ; Fetal Blood ; cytology ; Hematopoiesis ; physiology ; Humans ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Transplantation, Heterologous

OBJECTIVETo explore whether the co-transplantation of mesenchymal stem cells (MSC) from human umbilical cord blood (UCB) with UCB-derived CD34(+) cells in NOD/SCID mice could promote engraftment and accelerate hematopoiesis recovery.

METHODSAfter sublethal irradiated ((60)Co 2.5 Gy), NOD/SCID mice received within 24 hours UCB CD34(+) cells (1 x 10(5) per mouse for low dosage group or 1 x 10(6) per mouse for high dosage group) with or without human UCB-derived MSC (1 x 10(6) per mouse) transplantation by lateral tail vein injection. Peripheral blood cells of transplanted mice were measured for white blood cell count, hemoglobin and platelet count at 10th, 20th, 30th, 40th and 56th day. At the end of 8th week after transplantation, all the alive mice were sacrificed and human derived CD45(+), CD45(+)CD3(+), CD45(+)CD19(+), CD45(+)CD33(+) cells in the bone marrow (BM) were assayed by flow cytometry.

RESULTS(1) In the low dosage group, co-transplantation of MSC significantly raised the engraftment rate (26.02% vs 16.52%) (P < 0.05). (2) The survival rate in high dosage group was 80% for co-transplantation mice and 70% for CD34(+) cells alone transplantation mice. The survival rate in low dosage group was 70% for co-transplantation mice and 50% in CD34(+) cells transplantation mice. (3) In both dosages groups co-transplantation accelerated the hematopoiesis recovery. (4) At the end of 8 weeks after transplantation, in low dosage group, CD45(+)CD33(+) and CD45(+)CD19(+) cells were more in co-transplantation mice than in CD34(+) cells alone transplantation mice, but in high dosage group, the percentage of these two kinds of cells had no difference. In both dosage groups the percentage of CD45(+)CD41a(+) cells were higher in co-transplantation than in transplantation alone mice. CD45(+)CD3(+) cells were low in all groups.

CONCLUSIONS(1) In low dosage transplantation, human UCB MSC could promote human CD34(+) cells engraftment in transplanted mice. (2) Co-transplantation of human UCB MSC and human UCB CD34(+) cells could significantly promote the hematopoiesis reconstitution and improve the survival rate of NOD/SCID mice. (3) MSC could promote human UCB CD34(+) cells to differentiated into B-lymphocytes, granulocyte and megakaryocyte in vivo.

Animals ; Antigens, CD34 ; Cord Blood Stem Cell Transplantation ; Female ; Fetal Blood ; cytology ; Hematopoiesis ; Humans ; Mesenchymal Stem Cell Transplantation ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Transplantation, Heterologous

The study was aimed to explore the distribution and interaction mechanism of human bone marrow mesenchymal stem cells (MSC) and cord blood cytokine-induced killer (CIK)/natural killer (NK) cells infused via different ways at different times in NOD/SCID mice. 5 microl 1, 1'-dioctadecyl-3, 3, 3', 3'-tetramethylindocarbocyanine perchlorate (DiI) dye(red) was added in suspension of MSC per ml, and 1 microl carboxyfluorescein diacetate, succinimidyl ester(CFDA SE) dye(green) was added in suspension of CIK/NK cells per ml. The amounts of MSC and CIK/NK cells infused in each 6 NOD/SCID mouse were 1 x 10(6) (0.1 ml) and 1 x 10(7) (0.1 ml) respectively. All mice were divided into 4 groups, each group consisted of 6 mice. Group A: MSC (intravenous infusion, iv) + CIK/NK cells (iv) at the same time, group B: MSC (iv) + CIK/NK cells (iv) at 48 hours after infusion of MSC; group C: MSC (intramedullary infusion, im) + CIK/NK cells (iv) at the same time; group D: MSC (im) + CIK/NK cells (iv) at 48 hours after infusion of MSC. 3 NOD/SCID mice were sacrificed per batch at 24 hours and 48 hours after infused CIK/NK cells. Frozen sections of liver, spleen, lung and kidney were prepared, and then followed by counting the amounts of red and green fluorescence cells under fluorescence microscope, and calculating the ratio of MSC to CIK/NK cells for reflecting the interaction of MSC and CIK/NK cells in mice, and for showing the suppressive intensity of MSCs on CIK/NK cells. The results showed that the sums of average ratios of MSC to CIK/NK cells in lung, liver and spleen of group A and B were higher than that in group C and D at 24 hours and 48 hours respectively after infusing CIK/NK cells. The sum of average ratios of MSC to CIK/NK cells in group A was slightly higher than that in group B at 24 hours and 48 hours after infusing CIK/NK cells, but there was no significant difference between them. The sum of average ratios of MSC to CIK/NK cells in lung, liver and spleen in group C was slightly lower than that in group D at 24 hours after infusing CIK/NK cells, but reversed at 48 hours later and there was no significant difference between them. The sums of average ratios of MSC to CIK/NK cells in lung, liver and spleen in group A, B, C and D were all higher than those in kidney at 24 and 48 hours respectively after infusing CIK/NK cells. It is concluded, the MSC and CIK/NK cells may interact if they are infused via the same way and at the same time, the location where the suppression of MSC on CIK/NK cells occur in vivo may be reticulo-endothelial systems in lungs and livers.
Animals ; Bone Marrow Transplantation ; Cell Communication ; Cytokine-Induced Killer Cells ; transplantation ; Fetal Blood ; cytology ; Humans ; Killer Cells, Natural ; transplantation ; Liver ; cytology ; Lung ; cytology ; Mesenchymal Stem Cell Transplantation ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Transplantation, Heterologous

Bronchopulmonary dysplasia (BPD), a chronic lung disease affecting very premature infants, is a major cause of mortality and long-term morbidities despite of current progress in neonatal intensive care medicine. Though there has not been any effective treatment or preventive strategy for BPD, recent stem cell research seems to support the assumption that stem cell therapy could be a promising and novel therapeutic modality for attenuating BPD severity. This review summarizes the recent advances in stem cell research for treating BPD. In particular, we focused on the preclinical data about stem cell transplantation to improve the lung injury using animal models of neonatal BPD. These translational research provided the data related with the safety issue, optimal type of stem cells, optimal timing, route, and dose of cell transplantation, and potency marker of cells as a therapeutic agent. Those are essential subjects for the approval and clinical translation. In addition, the successful phase I clinical trial results of stem cell therapies for BPD are also discussed.
Bronchopulmonary Dysplasia/*therapy ; Cell- and Tissue-Based Therapy ; Clinical Trials as Topic ; Fetal Blood/cytology/transplantation ; Humans ; Infant, Newborn ; Infant, Premature ; *Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells/cytology

OBJECTIVETo investigate the feasibility of transplanting endothelial progenitor cells (EPCs) transfected with VEGF165 gene to free transplanted fat tissue for increasing neovascularization and the survival.

METHODSEPCs isolated from human cord blood were cultured in vitro and identified by immunocytochemistry. After transfection by VEGF165 gene, the expression of VEGF was assessed using ELISA. Then EPCs with (VEGF gene transfection group) and without VEGF165 gene transfection (EPCs group) were transplanted to free transplanted fat tissue at 18 nude mice's back, and nine nude mice transplanted with free fat tissue were injected with M199 (control group). CM-DiI was used to trace the transplanted cells. The capillary density of transplanted fat tissue was detected by CD34 immunohistochemistry.

RESULTSEPCs expressed cell markers CD34, KDR and CD133. After transfection, the expression of VEGF was positive. Transplanted EPCs survived and proliferated, and transplanted EPCs were incorporated into the capillary networks in the transplanted fat tissue. The percent of survival volume of transplanted fat tissue of VEGF gene transfection group was (96.2 +/- 8.6)%, significantly higher than that of the EPCs group [(75.3 +/- 6.8)%, P < 0.05) and M199 group [(40.2 +/- 2.5)%, P < 0.05). The capillary density of transplanted fat tissue of VEGF gene transfection group was significantly higher than those of the EPCs group and M199 group (P < 0.05).

CONCLUSIONSEPCs from human cord blood can increase free transplanted fat tissue neovascularization and the survival volume, and the ability of promoting neovascularization of EPCs transfected with VEGF165 gene is more potent than EPCs alone.

Adipose Tissue ; transplantation ; Animals ; Cord Blood Stem Cell Transplantation ; Endothelial Cells ; cytology ; physiology ; Female ; Fetal Blood ; cytology ; Graft Survival ; Humans ; Mice ; Mice, Nude ; Stem Cells ; cytology ; physiology ; Transfection ; Transplantation, Heterologous ; Vascular Endothelial Growth Factor A ; biosynthesis ; genetics ; physiology

OBJECTIVETo evaluate the hematopoietic reconstitution of implanted NOD/SCID mice, after intra-bone marrow cavity injection (iBM) of human umbilical cord blood (CB) mononuclear cells (MNCs).

METHODS24 female NOD/SCID mice were divided into different MNCs dosage iBM groups (3 x 10(6), 1 x 10(7), 3 x 10(7) cells), tail vein intravenous injection (iTV) group (3 x 10(7) cells) and control group (iBM of medium only). CB MNCs sorted by Ficoll-Hypaque were transplanted into left tibia bone marrow cavity of 6- to 8-week-old NOD/SCID mice, which were anesthetized and sublethally irradiated (270 cGy (137)Cs-gamma irradiation). The distribution of injected CB MNCs in noninjected right tibia of the same implanted mice was observed 24 hours after iBM. The establishment of hematopoiesis and the survival of mice were observed. BM cell surface CD marker expressions, dye Dil-CM tracing and human beta-actin from implanted mice were assessed 8 weeks after iBM or iTV.

RESULTSDil-CM marker could be detected on BM cells from noninjected right tibia 24 hours after iBM. Fourteen engrafted mice survived at the end of our study. Among them two, four and five were of iBM-1, iBM-2 and iBM-3 groups respectively, and one of control group and two of iTV group. White blood cell reconstitution was better in iBM mice than in iTV and control mice. There were human markers including CD45, Dil-CM and beta-actin DNA in the marrow cells from the human CB MNC engrafted mice.

CONCLUSIONThe preliminary results showed that hematopoiesis reconstitution by iBM was significantly better than iTV.

Animals ; Cells, Cultured ; Cord Blood Stem Cell Transplantation ; methods ; Female ; Fetal Blood ; cytology ; Humans ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Random Allocation ; Transplantation, Heterologous

OBJECTIVETo explore whether intra-bone marrow injection strategy could promote the engraftment of human umbilical cord blood derived hematopoietic stem/progenitor cells (HS/PC) in xenotransplanted NOD/SCID mouse model.

METHODSAliquots containing 1 x 10(3), 1 x 10(4), 0.5 x 10(5), 1 x 10(5) and 5 x 10(5) human umbilical cord blood (hUCB) CD34+ cells were transplanted into sublethally irradiated NOD/SCID mice via intra-venous (i.v.) and intra-bone marrow (iBM) injection. The homing and long-term engraftment capabilities of hUCB CD34+ cells from right tibia, right femur, left tibia, left femur and spleen were detected by PCR 24h after xenotransplantation and by FACS 8-week after xenotransplantation.

RESULTSTissues of liver, spleen, lungs, or cells from peripheral blood, right tibia, right femur, left tibia and left femur 24 hours after xenotransplantation in iBM injecting 5 x 10(5) CD34+ cells recipients expressed human chromosome 17 specific alpha-satellite fragment. 8-week engraftment of human cells was observed and engraftment level indicated dose-dependent effect in injected bone (right tibia) as well as non-injected bones (including right femur, left tibia and left femur), spleen and peripheral blood in all iBM recipients. 8-week engraftment levels of human cells were (44.063 +/- 20.095)% and (45.881 +/- 22.316)% for i.v. and iBM groups respectively, when transplanted with 1 x 10(5) hUCB CD34+ cells, being no statistical difference (P >0.05). More superior 8-week engraftment levels of human cells were observed in iBM recipients [(54.019 +/- 31.338)%] than in i.v. recipients [(12.197 +/- 10.350)%] when transplanted with 1.0 x 10(4) CD34 cells (P<0.01). Human cell engraftment was observed in iBM but not in i.v. recipients when transplanted with 1.0 x 10(3) CD34+ cells, and was usually observed in non-injected bones.

CONCLUSIONIntra-bone marrow strategy can efficiently increase the engraftment of umbilical cord blood derived hematopoietic stem/progenitor cells in xenotransplanted NOD/SCID mouse model.

Animals ; Antigens, CD34 ; Bone Marrow ; Cord Blood Stem Cell Transplantation ; methods ; Female ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Transplantation Conditioning ; Transplantation, Heterologous