Insensitive to PTH of CD8+ T cells regulate bone marrow mesenchymal stromal cell in aplastic anemia patients.

Aplastic anemia (AA) is a rare disorder characterized by the suppression of bone marrow function resulting in progressive pancytopenia. The pathogenesis of AA is complex and involves an abnormal hematopoietic microenvironment, hematopoietic stem cell/progenitor cell deficiencies, and immunity disorders. However, the underlying mechanism of the disease is still not fully uncovered. In this research, we collected both donor and patient samples and found suppressed proliferation, abnormal differentiation as well as increased apoptosis of patient mesenchymal stem cells (MSCs). Considering the close relationship of parathyroid hormone (PTH) and MSCs differentiation, further studies showed that although patients maintained normal serum PTH level, their CD8+ T cells possessed lower PTH receptors. The insensitive to PTH of patients' CD8+ T cells finally lead to reduced expression of key Wnt factors. In all, bone marrow CD8+ T cells may play an important role in inducing MSCs adipogenesis and osteogenesis imbalancement.

Secondary monoclonal gammopathy of undetermined significance is frequently associated with high response rate and superior survival in patients with plasma cell dyscrasias.

Secondary monoclonal gammopathy of undetermined significance (MGUS) is a special phenomenon that occurs during the treatment of multiple myeloma (MM). The incidence, biological characteristics, and prognostic value of secondary MGUS in patients with MM remain undefined. We proceed with a retrospective systematic review of serum immunofixation electrophoresis studies performed in 438 cases of patients with plasma cell dyscrasias, including 409 cases of newly diagnosed MM and 29 cases of primary plasma cell leukemia. Secondary MGUS was more common in patients with myeloma who had undergone stem cell transplantation than in those who had not (17 [29.8%] of 57 versus 5 [1.4%] of 352, P < .001). The clinical parameters and cytogenetic characteristics in patients with or without secondary MGUS were comparable. The complete response rates in patients with or without secondary MGUS were 81.8% and 21.8% respectively (P < .01). For the cohort as a whole, secondary MGUS was associated with significantly prolonged progression-free survival (median, 52.0 months versus 22.5 months; P = .002) and overall survival (median, not reached versus 35.0 months; P < .001). The presence of secondary MGUS retained independent prognostic value with a moderate impact on overall survival (hazard ratio .128 [95% confidence interval .018 to .922]; P = .041) in the multivariate Cox regression model. However, when analysis was restricted to patients undergoing stem cell transplantation, no statistical differences in progression-free survival and overall survival were found. In conclusion, we observe that secondary MGUS was frequently observed in MM patients after transplantation and conferred a survival prolongation. The favorable survival in patients with secondary MGUS may be explained by beneficial effect from myeloablative therapy.

A pivotal role of bone remodeling in granulocyte colony stimulating factor induced hematopoietic stem/progenitor cells mobilization.

The majority of hematopoietic stem/progenitor cells (HSPCs) reside in bone marrow (BM) surrounded by a specialized environment, which governs HSPC function. Here we investigated the potential role of bone remodeling cells (osteoblasts and osteoclasts) in homeostasis and stress-induced HSPC mobilization. Peripheral blood (PB) and BM in steady/mobilized state were collected from healthy donors undergoing allogeneic transplantation and from mice treated with granulocyte colony stimulating factor (G-CSF), parathyroid hormone (PTH), or receptor activator of nuclear factor kappa-B ligand (RANKL). The number and the functional markers of osteoblasts and osteoclasts were checked by a series of experiments. Our data showed that the number of CD45(-) Ter119(-) osteopontin (OPN)(+) osteoblasts was significantly reduced from 4,085 ± 135 cells/femur on Day 0 to 1,032 ± 55 cells/femur on Day 5 in mice (P = 0.02) and from 21.38 ± 0.66 on Day 0 to 14.78 ± 0.65 on Day 5 in healthy donors (P < 0.01). Decrease of osteoblast number leads to reduced level of HSPC mobilization regulators stromal cell-derived factor-1 (SDF-1), stem cell factor (SCF), and OPN. The osteoclast number at bone surface (OC.N/B.s) was significantly increased from 1.53 ± 0.12 on Day 0 to 4.42 ± 0.46 on Day 5 (P < 0.01) in G-CSF-treated mice and from 0.88 ± 0.20 on Day 0 to 3.24 ± 0.31 on Day 5 (P < 0.01) in human. Serum TRACP-5b level showed a biphasic trend during G-CSF treatment. The ratio of osteoblasts number per bone surface (OB.N/B.s) to OC.N/B.s was changed after adding PTH plus RANKL during G-CSF treatment. In conclusion, short term G-CSF treatment leads to reduction of osteoblasts and stimulation of osteoclasts, and interrupting bone remodeling balance may contribute to HSPC mobilization.

Transplantation of healthy but not diabetic outgrowth endothelial cells could rescue ischemic myocardium in diabetic rabbits.

OBJECTIVE: There are two types of endothelial progenitor cell (EPC) in circulation, early EPC and outgrowth endothelial cell (OEC). Diabetes impairs the function of EPC, but it is not clear whether transplantation of OECs can rescue ischemic myocardium in diabetes. In this study, we compared the function of diabetic and healthy OECs in vitro. Then we administered diabetic and healthy OECs intramyocardially and compared their contribution to vasculogenesis in diabetic rabbits. METHODS: Outgrowth endothelial cells from diabetic and healthy rabbits were isolated and subjected to in vitro proliferation, tube-forming, angiogenic cytokine assays. Exogenous diabetic and healthy OECs were analyzed for therapeutic efficacy in an acute ischemia model of diabetic rabbits. LV function was assessed using echocardiography. The capillary density and fibrosis area were evaluated. MRNA expression of VEGF and bFGF was analyzed using relative realtime quantitive PCR. RESULTS: Proliferation, tube-forming, secretion of VEGF and bFGF of diabetic OECs were significantly reduced compared with healthy OECs. In diabetic rabbits, healthy OECs transplantation could increase capillary density and improve cardiac function, decrease fibrosis area compared with diabetic OEC and the control group. Real time PCR indicated that mRNA expression of VEGF and bFGF were augmented more in the healthy OEC group than those in the control and diabetic OEC groups. CONCLUSIONS: These findings suggest that diabetes impairs the function of OECs. Transplantation of healthy OECs may rescue the ischemic myocardium by neovasculogenesis and paracrine effect in diabetic rabbits. However, autologous transplantation of diabetic OEC could not enhance cardiac function.

[Effect of human umbilical cord mesenchymal stem cells on the CD34+ cells transplantation in NOD/SCID mice].

OBJECTIVE: To study the effect of human umbilical blood (UB) mesenchymal stem cells (MSC) on the CD34(+) cells transplantation in NOD/SCID Mice. METHODS: Umbilical blood CD34(+) cells (3.5 x 10(5) cells) alone or combined with umbilical cord MSC cells were transplanted into NOD/SCID mice that had been irradiated with (137)Cs (3.0 Gy) before transplantation. Changes in peripheral blood cells within 6 post-transplantation weeks were detected. The mice were sacrificed 6 weeks after transplantation. The human hematopoietic cells (hCD45(+)) and multi-lineage engraftment cells (CD3/CD19, CD33, CD14, CD61, and CD235a) in NOD/SCID recipients bone marrow, spleen, and peripheral blood were analyzed by flow cytometry. RESULTS: In the 3rd post-transplantation week, white blood cells (WBC), platelets (PLT), and red blood cells (RBC) began to increase in both two groups. In the 6th post-transplantation week, WBC and PLT counts in CD34(+) + MSC group reached peak levels and were significantly higher than CD34(+) alone group (P < 0.05), while RBC level was not significantly different between these two groups P > 0.05). hCD45(+) cell levels in bone marrow and peripheral blood were (42.66 +/- 2.57) % and (4.74 +/- 1.02) % in CD34(+) + MSC group, which were significantly higher than those in CD34(+) alone group [(25.27 +/- 1.67) % and (1.19 +/- 0.54) %, respectively, P = 0.006]. Also in the 6th post-transplantation week, the proportions of CD19(+), CD33(+), CD14(+), CD61(+), and CD235a(+) in CD34(+) + MSC group were significantly higher than those in CD34(+) alone group (P < 0.05), while the proportion of CD3(+) T lymphocyte in CD34(+) + MSC group was significantly lower than that in CD34(+) alone group (P = 0.003). The amplification of CD19(+) B lymphocyte was significantly higher than other blood cell lineages (P < 0.05). CONCLUSION: The co-transplantation of MSC cells and CD34(+) cells can promote hematopoietic stem cell transplantation and hematopoietic recovery in vivo.

Glioblastoma extracellular vesicles induce the tumour-promoting transformation of neural stem cells.

Recurrent glioblastomas are frequently found near subventricular zone (SVZ) areas of the brain where neural stem cells (NSCs) reside, and glioblastoma-derived extracellular vesicles (EVs) are reported to play important roles in tumour micro-environment, but the details are not clear. Here, we investigated the possibility that NSCs are involved in glioblastoma relapse mediated by glioblastoma-derived EVs. We studied changes to NSCs by adding glioblastoma-derived EVs into a culture system of NSCs, and found that NSCs differentiated into a type of tumour-promoting cell. These transformed cells had distinguished proliferation activity, a high migration rate, and clone-forming ability revealed by CCK-8, wound healing and soft agar clone formation assays, respectively. In vivo assays indicated that these cells could accelerate tumour formation by Ln229 cells in nude mice. Moreover, to explore the mechanisms underlying NSC transformation, single cell transcriptome sequencing was performed; our results suggest that several key genes such as S100B, CXCL14, EFEMP1, SCRG1, GLIPR1, HMGA1 and CD44 and dysregulated signalling may be important for the transformation of NSCs. It is also indicated that NSCs may be involved in glioblastoma recurrence through EV release by glioblastoma in this work. This could help to illuminate the mechanism of glioblastoma relapse, which occurs in a brief period after surgical excision, and contribute to finding new ways to treat this disease.

Repetitive and Prolonged Omega-3 Fatty Acid Treatment After Traumatic Brain Injury Enhances Long-Term Tissue Restoration and Cognitive Recovery.

Traumatic brain injury (TBI) is one of the most disabling clinical conditions that could lead to neurocognitive disorders in survivors. Our group and others previously reported that prophylactic enrichment of dietary omega-3 polyunsaturated fatty acids (n-3 PUFAs) markedly ameliorate cognitive deficits after TBI. However, it remains unclear whether a clinically relevant therapeutic regimen with n-3 PUFAs administered after TBI would still offer significant improvement of long-term cognitive recovery. In the present study, we employed the decline of spatial cognitive function as a main outcome after TBI to investigate the therapeutic efficacy of post-TBI n-3 PUFA treatment and the underlying mechanisms. Mice were subjected to sham operation or controlled cortical impact, followed by random assignment to receive the following four treatments: (1) vehicle control; (2) daily intraperitoneal injections of n-3 PUFAs for 2 weeks, beginning 2 h after TBI; (3) fish oil dietary supplementation throughout the study, beginning 1 day after TBI; or (4) combination of treatments (2) and (3). Spatial cognitive deficits and chronic brain tissue loss, as well as endogenous brain repair processes such as neurogenesis, angiogenesis, and oligodendrogenesis, were evaluated up to 35 days after TBI. The results revealed prominent spatial cognitive deficits and massive tissue loss caused by TBI. Among all mice receiving post-TBI n-3 PUFA treatments, the combined treatment of fish oil dietary supplement and n-3 PUFA injections demonstrated a reproducible beneficial effect in attenuating cognitive deficits although without reducing gross tissue loss. Mechanistically, the combined treatment promoted post-TBI restorative processes in the brain, including generation of immature neurons, microvessels, and oligodendrocytes, each of which was significantly correlated with the improved cognitive recovery. These results indicated that repetitive and prolonged n-3 PUFA treatments after TBI are capable of enhancing brain remodeling and could be developed as a potential therapy to treat TBI victims in the clinic.

Inhibition of Na+-K+-2Cl- cotransporter attenuates blood-brain-barrier disruption in a mouse model of traumatic brain injury.

Traumatic brain injury (TBI) can lead to long-term motor and cognitive dysfunction, which can be at least partly attributed to blood-brain barrier (BBB) disruption. The mechanisms underlying post-TBI BBB disruption, however, are poorly understood thus far. Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) is a universally expressed ion transporter that maintains intracellular ion homeostasis by increasing intracellular K+ and Cl-. Having been characterized in stroke models, NKCC1 is activated in various cell types in the ischemic brain, and is thought to mediate BBB disruption, brain edema, and neuronal cell death. In this study, we tested the hypothesis that inhibition of NKCC1 may improve neurological outcomes via protecting against BBB disruption in a TBI mouse model. Adult male C57BL/6 J mice or NKCC1 deficient mice were subjected to controlled cortical impact (CCI). As an alternative to the genetic-based NKCC1 depletion, bumetanide, a selective NKCC1 inhibitor, was administrated (25 mg/kg, i.p.) 15 min after CCI and then every 6 h up to 48 h. Short-term sensorimotor function recovery was determined by rotarod, cylinder test, grid walking and foot fault test. BBB integrity was examined at 48 h post-CCI by measuring Evans blue extravasation, brain water content, and expression levels of tight junction proteins. Our results revealed that administration of bumetanide or genetic depletion of NKCC1 improved short-term neurological recovery against TBI. Bumetanide treatment markedly decreased brain water content and BBB leakage, correlated with reduction of MMP-9 expression and preventing the degradation of tight junction proteins. These findings suggest an important role of NKCC1 activation in mediating BBB disruption after TBI. Thus, NKCC1 inhibition may offer the potential for improving neurological outcomes in clinical TBI.

Co-delivery of tumor-derived exosomes with alpha-galactosylceramide on dendritic cell-based immunotherapy for glioblastoma.

Dendritic cell (DC) vaccine-based immunotherapy for glioblastoma multiforme (GBM) has shown apparent benefit in animal experiments and early-phase clinical trials, but the survival benefit is variable. In this work, we analyzed the mechanism of the potent antitumor immune response induced in vivo by tumor-associated antigen (TAA)-specific DCs with an invariant natural killer T (iNKT) cell adjuvant in orthotopic glioblastoma-bearing rats vaccinated with tumor-derived exosomes and α-galactosylceramide (α-GalCer) -pulsed DCs. Compared with traditional tumor lysate, exosomes were utilized as a more potent antigen to load DCs. iNKT cells, as an effective cellular adjuvant activated by α-GalCer, strengthened TAA presentation through their interaction with DCs. Co-delivery of tumor-derived exosomes with α-GalCer on a DC-based vaccine showed powerful effects in glioblastoma immunotherapy. This vaccine induced strong activation and proliferation of tumor-specific cytotoxic T lymphocytes, synergistically breaking the immune tolerance and improving the immunosuppressive environment.

[The effect of matrix metalloproteinase-9 in granulocyte colony stimulation factor-induced stem cell mobilization].

OBJECTIVE: To investigate the effects of matrix metalloproteinase-9 (MMP-9) on granulocyte colony stimulation factor (G-CSF)-induced hematopoietic stem/progenitor cell (HSPC) mobilization in healthy donors of hematopoietic stem cells. METHODS: Peripheral blood (PB) samples and bone marrow (BM) blood samples were collected from 12 healthy donors of hematopoietic stem cell before and 5 days after G-CSF-induced mobilization. CD34(+) cells were isolated and purified. ELISA was used to detect the protein expression of MMP-9 in the peripheral blood and BM blood of the healthy donors. The protein expression of MMP-9 in the BM blood was detected by ELISA and immunohistochemistry, and the stromal cell-derived factor-1 (SDF-1) level in the BM blood was detected by ELISA. The mRNA expression of MMP-9 in the BM blood samples was detected by RT-PCR. HT1080 cells rich in MMP-9 were cultured. CD34(+) cells were co-cultured with the supernatant of HT1080 cell culture fluid. CD34(+) cells cultured in Iscove's modified Dulbecco's medium were used as control group. Fluorescence-activated cell sorter was used to detect the CXCR4 expression on the surface of the CD34(+) cells. In the transwell experiment CD34(+) cells were divided into 4 groups: control group, o-phenanthroline (MMP-9 chemical inhibitor, MPI) group, HT1080 sup group, and HT1080 + MPI group to be co-cultured with buffer, o-phenanthroline, supernatant of culture fluid of HT1080 cells, or supernatant of culture fluid of HT1080 cells Flow cytometry was used to calculate the cell migration capacity. RESULTS: The MMP-9 level of BM and PB of the healthy donors 5 days after G-CSF mobilization were 278 ng/ml +/- 34 ng/ml and 392 ng/ml +/- 284 ng/ml respectively, both significantly higher than those before G-CSF mobilization (42 ng/ml +/- 17 ng/ml and 27 ng/ml +/- 12 ng/ml respectively (P < 0.01 and P < 0.05). Western blotting showed that the SDF-1 level in the supernatant 5 days after G-CSF mobilization was 5.9 ng/ml +/- 1.0 ng/ml, significantly lower than that before G-CSF mobilization (7.2 ng/ml +/- 0.7 ng/ml, P < 0.05). The CXCR4 levels of the CD34(+) cell from both PB and BM blood were up-regulated after co-culture with the supernatant of HT1080 cells (both P < 0.05). The migration capacity of CD34(+) cells cultured in the supernatant of HT1080 cells was increased significantly (P < 0.05), however, this effect could be inhibited by MIP (P < 0.05). The PB WBC numbers of the G-CSF group and G-CSF + MPI group were 14.9 x 10(6)/L +/- 4.3 x 10(6)/L and 12.3 x 10(6)/L +/- 1.2 x 10(6)/L respectively, the PB WBC numbers of the G-CSF + MPI group was significantly lower than that of the G-CSF group (P < 0.05), however, significantly higher than that of the negative control group (6.8 x 10(6)/L +/- 2.5 x 10(6)/L, P < 0.05). The CFU of the G-CSF group was (84 +/- 10) U/2 x 10(5) MNC, significantly higher than that of the G-CSF + MPI group, (69 +/- 3) U/2 x 10(5) MNC (P < 0.05). The BM MNC number of the G-CSF group was 12.7 x 10(6)/L +/- 0.7 x 10(6)/L, not significantly different from that of the G-CSF + MPI groups (13.1 x 10(6)/L +/- 1.3 x 10(6)/L; P > 0.05). CONCLUSION: MMP-9 probably facilitates HSPC mobilization by degrading SDF-1, up-regulating CXCR4 expression on the CD34(+) cells, and increasing the migration ability of CD34(+) cells.

[The expression and clinical significance of early differentiation antigens in acute leukemia].

OBJECTIVE: To evaluate the expression and clinical significance of early differentiation antigens of hematopoietic cells CD(34), CD(90) and CD(133) in acute leukemia (AL). METHODS: The expression of CD(34), CD(90) and CD(133) on leukemic blasts in 76 AL patients was detected with three-color direct flow-cytometry and CD(133) mRNA was detected with hemi-quantitative reverse transcriptive polymerase chain reaction (RT-PCR). RESULTS: (1) CD(34) and CD(133) expression in AL patients was significantly higher than that in controls (46.37% vs 0.47%, 21.93% vs 0.29%, P < 0.01), but there was no obvious difference of CD(90) expression between them (0.51% vs 0.25%, P > 0.05). The expression of CD(133) antigen was highly correlated with CD(133) mRNA expression in both of the normal control donors and AL patients (r = 0.932, P < 0.01). (2) The positive rates of CD(34), CD(90) and CD(133) in all AL patients were 63.2%, 7.9% and 42.1%, respectively. The expression of CD(90) in ALL was higher than that in AML (P < 0.05). Positive expression of CD(133) in AML-M(4) was significantly higher than that in other AML subtypes (P < 0.01). The positive rate of CD(34) in B-ALL was much higher than that in T-ALL (P < 0.05). (3) CD(133) expression in AML was significantly correlated with the expression of CD(34) and HLA-DR (P < 0.01). (4) The expression of CD(34), CD(90) and CD(133) was not associated with the clinical prognostic factors such as cytogenetic or molecular aberrations, initial peripheral blood WBC counts, lactate dehydrogenase level, multiple drug resistant expression and age. (5) There was a trend toward lower completely remission (CR) rate and overall survival rate in CD(34), CD(90) and CD(133) positive cases, but only CD(34)(+)/CD(133)(+) cases had significant lower CR rate than negative ones (P < 0.05). CONCLUSIONS: It is indicated AL has significantly higher CD(34) and CD(133) expression as compared with the normal controls. CD(133)/CD(34) co-expression might provide adverse prognostic stratification of acute leukemia.

Targeting stem cell niche can protect hematopoietic stem cells from chemotherapy and G-CSF treatment.

INTRODUCTION: Hematopoietic stem/progenitor cells (HSPCs) reside in a tightly controlled local microenvironment called bone marrow niche. The specialized microenvironment or niche not only provides a favorable habitat for HSPC maintenance and development but also governs stem cell function. METHOD: We investigated the effect of cytotoxic drugs on bone marrow niche. To mimic the multiple rounds of chemotherapy followed by autologous hematopoietic stem cells (HSCs) transplantation in a clinical setting, we further verified the hypothesis that targeting the niche might improve stem cell-based therapies in mouse models. RESULTS: We found that multiple rounds of cytotoxic drug treatment significantly disrupted niche and serum osteocalcin level was significantly reduced after treatment in autologous HSPCs transplanted patients (P = 0.01). In mouse models, the number of CD45(-)Ter119(-)OPN(+) osteoblasts was significantly reduced after multiple rounds of chemotherapies and granulocyte colony stimulating factor (G-CSF) treatment (P < 0.01). Parathyroid hormone (PTH) or receptor activator of nuclear factor kappa-B ligand (RANKL) treatment significantly increased the number of HSCs mobilized into peripheral blood (PB) for stem cell harvesting and protected stem cells from repeated exposure to cytotoxic chemotherapy. Treatments with G-CSF and PTH significantly increased the preservation of the HSC pool (P < 0.05). Moreover, recipient mice transplanted with circulation HSPCs that were previously treated with PTH and RANKL showed robust myeloid and lymphatic cell engraftment compared to the mice transplanted with HSCs after chemotherapy or G-CSF treatment. CONCLUSION: These data provide new evidence that the niche may be an important target for drug-based stem cell therapy.

[The effects of the short-term ex vivo expansion on the adhesion characteristics and chemotactic function of expanded ex vivo CD34(+) cells].

OBJECTIVE: To study the effect of ex vivo expansion on the adhesion activities and chemotactic function of umbilical cord blood (UCB) hematopoietic stem and progenitor cells (HSPCs). METHODS: CD34(+) cells isolated from fresh UCB samples were cultured in serum-free and stroma-free culture system. After 7, 10 and 14 days' culture, CD34(+) cells were re-selected from the expanded products. Stromal cell- derived factor-1 (SDF-1) 100 ng/ml was added into the experimental CD34(+) cells and the absorbance at 570 nm of all groups was examined. 20 micro g/ml fibronectin (Fn) was added and the spontaneous adhesion between CD34(+) and FN was detected by MTT method. The homing-related functions including expression of homing-related adhesion molecules (CAM), adhesion activity and chemotactic function of the re-selected CD34(+) cells were evaluated and compared with those of the initial fresh CD34(+) cells. RESULTS: (1) The expression of CD49d, CD44, CD11a and CD49e on expanded CD34(+) cells increased or sustained the same levels as those of the fresh isolated UCB CD34(+) cells, while the expression of CD62L, CD54 and CD31 on expanded CD34(+) cells declined during the culture. (2) The spontaneous adhesion between CD34(+) and FN and SDF-1-induced adhesion continuously increased in the course of the first 10-day culture. The spontaneous adhesion rate and SDF-1-induced adhesion rate on day 0, day 7 and day 10 were 28% and 63%, 60% and 70%, 63% and 90% respectively. (3) The migration efficiency of re-selected CD34(+) cells on day 7 was almost the same compared to that of fresh CD34(+) cells. CONCLUSION: The expanded HSPCs sustain most of the homing-related characteristics and activities during one-week culture while extended culture may partly impair their intrinsic homing potential.

Bone marrow stromal cells protect myeloma cells from bortezomib induced apoptosis by suppressing microRNA-15a expression.

Despite unsurpassed anti-tumor activity of bortezomib for multiple myeloma (MM), drug resistance has emerged as a challenge, especially when MM cells adhere to the stroma. This study aimed to determine whether bone marrow stromal cells (BMSCs) have a role in the development of chemoresistance in MM. Our data demonstrate that the secretion of interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), and cell-to-cell contact with microenvironment-derived stromal cells from patients with multiple myeloma (MM-BMSCs) significantly decreased the sensitivity of myeloma cells to bortezomib treatment. Mechanistically, we found that microRNA (miRNA)- 15a expression was up-regulated in U266 and NCI-H929 cells treated by bortezomib, which was inhibited by MM-BMSCs. miRNA-15a transfected myeloma cells were arrested in G1/S checkpoint and secreted less VEGF compared to control transfected cells, although no significant difference was found in VEGF mRNA levels. In conclusion, our data suggest that via suppressing miRNA-15a expression, BMSCs provide survival support and protect myeloma cells from bortezomib induced apoptosis.

Umbilical cord-derived mesenchymal stem cells isolated by a novel explantation technique can differentiate into functional endothelial cells and promote revascularization.

Stem cells transplantation holds great promise for the treatment of ischemic diseases through functional revascularization. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) are also an ideal candidate for cell-based bioengineering. Herein, we report on the development of a simple and effective protocol to isolate UC-MSCs, and confirm their endothelial potential both in vitro and in vivo. UC-MSCs were isolated by a novel explantation technique and induced to differentiate into endothelial-like cells. Then UC-MSCs were transplanted into ischemic mouse model and cultured on 3D gel/MMT-CS composite scaffolds. Morphological and proliferation assessments show that sufficient UC-MSCs can be generated during a relatively short culture period with explantation technique. Increased expression of endothelial-specific markers (KDR and vWF), and functional markers (ac-LDL uptake and UEA-1 binding), indicate that functional endothelial progenitor cells are induced after 9 days of in vitro culture. In an ischemic hindlimb mouse model, the ratio of ischemic/nonischemic limb perfusion 4 weeks after MSCs transplantation reached 0.84 +/- 0.09. The capillary density of this group was 2.57-fold greater than that of sham-injected mice (P < 0.05). Immunofluorescence and immunohistological analyses indicate that MSCs may act to salvage the ischemic tissue by incorporating into the local vasculature. In vitro, UC-MSCs were observed to incorporate into 3D gel/MMT-CS composite scaffolds, to secrete extracellular matrix, to remain viable, and to retain their proliferation capacity. In conclusion, UC-MSCs isolated by novel yet simple explantation technique are well suited for application in the development of novel stem cell-based revascularization therapies.

[Effect of mesenchymal stem cells on multiple myeloma cells growth and inhibition of bortezomib induced cell apoptosis].

OBJECTIVE: To investigate the role of mesenchymal stem cells (BMSCs) in multiple myeloma (MM) bone marrow (BM) microenrivonment and their effect on myeloma cells survival and bortezomib induced apoptosis. METHODS: BMSCs were derived from BM of untreated myeloma patients (MM-BMSCs) and healthy donors (HD-BMSCs), respectively. The phenotype, proliferation time and cytokine secretion of MM-BMSCs were detected and compared with HD-BMSCs. Then BMSCs were co-cultured with myeloma cell line NCI-H929 and bortezomib in vitro. The NCI-H929 cells proliferation and bortezomib induced cell apoptosis were investigated. RESULTS: MM-BMSCs and HD-BMSCs were isolated successfully. The phenotype of MM-BMSCs was similar to that of HD-BMSCs. Expressions of CD73, CD105, CD44 and CD29 were positive, but those of CD31, CD34, CD45 and HLA-DR (< 1%) negative. The proliferation time of MM-BMSCs was longer than that of HD-BMSCs (82 h vs 62 h, P < 0.05). Moreover, over-expressions of IL-6 and VEGF in MM-BMSCs culture supernatant were detected as compared with that in HD-BMSCs [(188.8 ± 9.4) pg/ml vs (115.0 ± 15.1) pg/ml and (1497.2 ± 39.7) pg/ml vs (1329.0 ± 21.1) pg/ml, respectively]. MM- BMSCs supported survival of the myeloma cells NCI-H929 and protected them from bortezomib induced cell apoptosis. CONCLUSIONS: MM-BMSCs is benefit for myeloma cells proliferation and against cell apoptosis induced by bortezomib. Over-expression of IL-6 and VEGF maybe play a critical role in these effects.

B7-H1 up-regulation on dendritic-like leukemia cells suppresses T cell immune function through modulation of IL-10/IL-12 production and generation of Treg cells.

Dendritic-like leukemia cells (DLLC) originating from leukemic cells could potentially induce a T cell-mediated anti-leukemia immune response. It has been demonstrated that B7-H1, a newly identified homologue of CD80/CD86, is abundant in human carcinomas and dendritic cells (DC), can exert co-stimulatory and immune regulatory functions. We demonstrated that B7-H1 was significantly expressed on AML cells and was strongly enhanced after differentiation to DLLC. Blockade of B7-H1 expressed on DLLC results in increased T cell proliferation and Th1 cytokine production, and decreased Th2 cytokine production. Importantly, autologous CTLs induced by DLLC treated with B7-H1 mAb showed significantly increased specific cytotoxcity against AML blasts. We further demonstrated that a significant decrease in IL-12 production, increase in IL-10 production by DLLC, and an increased CD4(+)CD25(+)Foxp3(+) T regulatory population lead to the defective T cell immune response that is induced by B7-H1 up-regulation on DLLC. Our data suggest that up-regulated B7-H1 on DLLC acts as a strong inhibitor of anti-leukemia T cell response, and that blockade of B7-H1 can improve DLLC-mediated anti-leukemia immunity.

[Study of influence of umbilical cord mesenchymal stem cells on CD34+ cells in vivo homing in NOD/SCID].

OBJECTIVE: To investigate the effect and the potential mechanism of umbilical cord (UC) derived mesenchymal stem cells (MSCs) on umbilical cord blood (UCB) derived CD34+ cells in vivo homing in xenotransplanted NOD/SCID mice model. METHODS: CD34+ cells and MSCs were derived from fresh UCB and UC, respectively. CD34+ cells (5 x 10(5) per mice) and MSC cells (5 x 10(6) per mice) were co-transplanted into irradiated NOD/SCID mice intravenously. CD34+ cells (5 x 10(5) per mice) alone were transplanted into the mice as control group. CD34+ cells home in bone marrow and spleen of recipient mice were detected 20 hours after transplant by FACS and RT-PCR, and the homing efficiencies were calculated. The effect of MSCs on CD34+ cells chemotactic function was investigated after co-cultured UCB CD34+ cells with UC MSCs in vitro. After 4 and 7 days coculture, the homing related adhesion molecules (the CD49e, CD31, CD62L, CD11a) expressed on CD34+ cells were detected by FACS. RESULTS: 1) The homing efficiencies in bone marrow in experimental and control group were (7.2 +/- 1.1)% and (5.4 +/- 0.9)%, respectively (P < 0.05). 2) Human GAPDH gene was detected in bone marrow in experimental group and in spleen in both groups. 3) The migration efficiency of CD34+ cells was significantly higher in experimental group (35.7 +/- 5.8)% than in control group (3.5 +/- 0.6)% (P < 0.05). 4) The expression of CD49e, CD31, CD62L on CD34+ cells kept higher level in MSCs cocultured group than in CD34+ cells alone group. CONCLUSIONS: MSCs can efficiently increase homing of CD34+ cells to bone marrow and spleen in vivo by keeping a high level of homing adhesion molecules expression and improving migration efficiency of UCB CD34+ cells.

[Differences in megakaryocyte progenitor ex vivo expansion between CD34+ cells derived from human umbilical cord blood and bone marrow].

The purpose of this study was to explore the differences in megakaryocyte progenitor ex vivo expansion between CD34+ cells derived from human umbilical cord blood (CB) and bone marrow (BM). Mononuclear cells (MNCs) were obtained from CB or BM by Ficoll-Hypaque density gradient separation. CD34+ cells were purified by magnetic cell sorting (MACS). The selected CD34+ cells were seeded in serum-free conditions stimulated with thrombopoietin (TPO), TPO+interleukin 11 (IL-11), or TPO+IL11+heparin for 14 days. Amplification product (CD34+, CD41a+, and CD34+ CD41a+ cells) immunophenotypes, megakaryocyte apoptosis rates and the DNA content were measured by fluorescence-activated cell sorting (FACS). The colony-forming units of granulocytes and monocytes (CFU-GM), burst-forming units of erythrocytes (BFU-E), and colony-forming units of megakaryocytes (CFU-Mk) were also evaluated by the colony-forming units (CFU) assay. The results indicated that CD34+ cells derived from CB showed higher expansion ability of total cell counts, CD41a+ and CD34+ CD41a+ cells than those derived from BM for all days 14 of culture (p<0.05, respectively). There were no significant differences in CFU-GM, BFU-E, and total CFU-Mk counts between CB and BM-derived CD34+ cells on day 0 (p>0.05, respectively), but CB-derived CFU-Mk seemed mainly large colonies, and the number of large colonies was higher than that from BM (p<0.05) on day 0. There were no significant differences in expansion ability of CFU-GM between CB and BM-derived cells on days 7, 10, and 14 of culture (p > 0.05, respectively), but the expansion ability of BFU-E and CFU-Mk derived from CB cells was higher than that from BM (p<0.05, respectively). There were no significant differences in apoptosis rates of megakaryocyte from two source cells for days 14 of culture. Megakaryocytes derived from CB mostly showed the 2N DNA content (>90%) for days 14 of culture, while those cells derived from BM showed the increased DNA content, and 4N, 8N or more ploidy cells gradually increased with prolonging of culture time. It is concluded that CB-derived CD34+ cells have a greater proliferation potential than that derived from BM, which is therefore proven to be a better cell source for megakaryocyte progenitor expansion in vitro.