Number Of Colony-forming Unit Fibroblasts Research Articles

Study On Mechanism of Bone Marrow Mesenchymal Stem Cell in Treating Patients with Rheumatoid Arthritis.

Abstract 4486 ObjectiveTo explore mechanism of Mesenchymal stem cells in treating Rheumatoid arthritis. Methods(1) MSCs were isolated from Bone marrow samples of Rheumatoid arthritis(RA)patients and purified by density gradient centrifagation and cultured in vitro. Morphology, immunophenotype, and proliferative property of bMSC and colony forming unit-fibroblast (CFU-F) were measured and analyzed. (2) In an in vitro co-culture system, MSCs were observed to modulate proliferation,activation, and maturation of T and B lymphocytes of Rheumatoid arthritis(RA)patients. The expression of IL-1?ATNF-a?ATGF-β were obviously changed.(3) Bone marrow- derived BMSCs of wistar rats were isolated and cultured in vitro routinely and the fourth passage as taken for identification of specific surface antigens by flowcytometry, then were labled with 5- BrdU in vitro. The model of collagen-induced arthritis (CIA) rats were established. 5- BrdU labled BMSCs were implanted through tail vein to model rats. At 4 weeks after BMSCs transplantation, immunohistochemical examinations were used to investigate BMSCs aggregate around the knee joints.and identify the contribution of bone marrow– derived cells to joints damage repair. Results(1) The culture expanded cells from RA patients presented a typical fibroblast-like morphology. Cells were positive for SH2(CD105),CD71,and CD44, but negative for CD45.Their proliferative capacity and CFU-F number were similar to those of bMSCs from healthy donors. (2) MSCs significantly inhibited T,B cell proliferation. MSCs also could down-regulating the levels of IL-1, TNF and up-regulating TGF-β. (3) Flow cytometry showed that BMSCs expressed CD44, CD71and CD105, but no CD45,CD34. At 4 weeks after the cells transplantation, the implanted cells were detected in the damaged joints of the model rats, which is not founded in normal knee joints of the rats'.and at same time there are more OPG(osteoprotegerin) positive. Conclusion(1) In the aspect of morphology, immuno -phenotypen, proliferative property and colony forming unit-fibroblast (CFU-F),MSCs from bone marrow of RA patients are not different from those of MSCs isolated from bone marrow of normal donors,MSCs from the bone marrow of RA patients have the potentiality in clinical application.(2) Human bone marrow MSCs inhibited Tcell and Bcell activation and proliferation in patients with RA in vitro. And these immuno –modulatory effects were not MHC-restricted. (3) bone marrow mesenchymal stem cells prevents tissue damage in arthritis. Allogeneic MSCs can engraft at sites of tissue damage,and prepair damage. That provided positive results for developing effective therapy for Rheumatoid arthritis. Disclosures:No relevant conflicts of interest to declare.

Obstructive Apneas Induce Early Release of Mesenchymal Stem Cells into Circulating Blood

To investigate whether noninvasive application of recurrent airway obstructions induces early release of mesenchymal stem cells into the circulating blood in a rat model of obstructive sleep apnea. Prospective controlled animal study. University laboratory. Twenty male Sprague-Dawley rats (250-300 g). A specially designed nasal mask was applied to the anesthetized rats. Ten rats were subjected to a pattern of recurrent obstructive apneas (60 per hour, lasting 15 seconds each) for 5 hours. Ten anesthetized rats were used as controls. Mesenchymal stem cells from the blood and bone marrow samples were isolated and cultured to count the total number of colony-forming unit fibroblasts (CFU-F) of adherent cells after 9 days in culture. The number of CFU-F from circulating blood was significantly (P = 0.02) higher in the rats subjected to recurrent obstructive apneas (5.00 +/- 1.16; mean +/- SEM) than in controls (1.70 +/- 0.72). No significant (P = 0.54) differences were observed in CFU-F from bone marrow. Application of a pattern of airway obstructions similar to those experienced by patients with sleep apnea induced an early mobilization of mesenchymal stem cells into circulating blood.

The investigation of using TRAIL in vitro to remove leukemia cells from the autologous hemopoietic stem cell transplants

Objective To explore the feasibihty of TRAIL to be used to remove the leukemia cells from the autologous hemopoietic stem cell transplants. Methods The expression of decoy receptor 1 and decoy receptor 2 on the bone marrow mononuclear cell were routine-ly isolated and observed by fluorescence microscope after PE-DcR1 or PE-DcR2 stain. The apeptosis rates of mononuclearcell and Jurkat cells interfered by 200ng/ml TRAIL for 18h were determined by flow cytometry after AnnexinV/Pl stain. The interfered mononuclear cells were cultured to count the number to form colony-forming unit-fibroblast(CFU-F). The Jurkat cells labeled by green fluorescent protein were in-corporated into the mononuclear ceils and affected by 200ng/ml TRAIL for 24h. The incorporated cells were cultured in the system with GM-CSF and EPO and the numbers of the CFU-GM, BFU-E and fluorescence colony were counted on the seventh day. Results Both decoy re-ceptor land decoy receptor 2 of TRAIL can be detected on membrane or in cytoplasm of the bone marrow mononuclear cell. The apoptosis rate of mononuclear cell interfered by 200ng/ml TRAIL for 18h was (5.95±1.23)%, which was markedly lower than that of Jurkat cells (33.42±2.28) %. The number of CFU-F of TRAIL group and control group were 235.67 ~ 33.56 and 249.33±42.72, respectively. No marked difference can be found between the mentioned two groups. Moreover, TRAIL decreased the number of fluorescence colony formed by Jurkat cells without significant decreasing the number of CFU-GM and BFU-E formed by bone marrow mononuclear cells. Conclusion TRAIL can selectively induce apoptosis in Jurkat cells without marked toxic effect on the bone marrow mononuclear cells, which means that TRAIL can be used to remove the leukemia cells from the autologous hemopeietic stem cell transplants in vitro. Key words: Tumor necrosis factor/PD ; Membrane glycoproteins/PD; Hematopoietic stem cell transplantation; Leukemia

Isolation and Characterization of Mouse Mesenchymal Stem Cells

Objective Mesenchymal stem cells (MSCs) have been studied in regenerative medicine because of their unique immunologic characteristics. However, before clinical application in humans, animal models are needed to confirm their safety and efficacy. To date, appropriate methods and sources to obtain mouse MSCs have not been identified. Therefore, we investigated MSCs isolated from 3 strains of mice and 3 sources for the development of MSCs in a mouse model. Materials and Methods Male BALB/c, C3H and C57BL/6 mice were used to isolate MSCs from various tissues including bone marrow (BM), compact bone, and adipose tissue. The MSCs were maintained in StemXVivo medium. Immunophenotypes of the MSCs were analyzed by FACS and their growth potential estimated by the number of colony-forming unit fibroblasts. Results All MSCs that were isolated from BM, compact bone, and adipose tissue showed plastic-adherent, fibroblastic-like morphologic characteristics regardless of the mouse strain or cell source. However, culture of BM MSCs was less successful than the other tissue types. The FACS phenotype analysis revealed that the MSCs were positive for CD29, CD44, CD105, and Sca-1, but negative for CD34, TER-119, CD45, and CD11b. According to the results of the characterization, the adipose tissue MSCs showed higher growth potential than did other MSCs. Conclusion The results of this study showed that culture of adipose tissue and compact bone-MSCs was easier than BM MSCs. Based on the results of immunophenotype and growth potential, C57BL/6 AT-MSCs might be a suitable source to establish a mouse model of MSCs.

Human placenta and bone marrow derived MSC cultured in serum-free, b-FGF-containing medium express cell surface frizzled-9 and SSEA-4 and give rise to multilineage differentiation

Conventionally, mesenchymal stem cells (MSC) are generated by plating cells from bone marrow (BM) or other sources into culture flasks and selecting plastic-adherent cells with fibroblastoid morphology. These cells express CD9, CD10, CD13, CD73, CD105, CD166, and other markers but show only a weak or no expression of the embryonic markers stage-specific embryonic antigen-4 (SSEA-4), Oct-4 and nanog-3. Using a novel protocol we prepared MSC from BM and non-amniotic placenta (PL) by culture of Ficoll-selected cells in gelatin-coated flasks in the presence of a serum-free, basic fibroblast growth factor (b-FGF)-containing medium that was originally designed for the expansion of human embryonic stem cells (ESC). MSC generated in gelatin-coated flasks in the presence of ESC medium revealed a four-to fivefold higher proliferation rate than conventionally prepared MSC which were grown in uncoated flasks in serum-containing medium. In contrast, the colony forming unit fibroblast number was only 1.5- to twofold increased in PL-MSC and not affected in BM-MSC. PL-MSC grown in ESC medium showed an increased surface expression of SSEA-4 and frizzled-9 (FZD-9), an increased Oct-4 and nestin mRNA expression, and an induced expression of nanog-3. BM-MSC showed an induced expression of FZD-9, nanog-3, and Oct-4. In contrast to PL-MSC, only BM-MSC expressed the MSC-specific W8B2 antigen. When cultured under appropriate conditions, these MSC gave rise to functional adipocytes and osteoblast-like cells (mesoderm), glucagon and insulin expressing pancreatic-like cells (endoderm), as well as cells expressing the neuronal markers neuron-specific enolase, glutamic acid decarboxylase-67 (GAD), or class III β-tubulin, and the astrocyte marker glial fibrillary acidic protein (ectoderm). In conclusion, using a novel protocol we demonstrate that adult BM-and neonatal PL-derived MSC can be induced to express high levels of FZD-9, Oct-4, nanog-3, and nestin and are able of multi-lineage differentiation.

Expansion and Multipotencial Differentiation of Mesenchymal Stem Cells Isolated from Patients after High Dose Chemotherapy.

Background. High dose chemotherapy (HDCT) followed by autologous PBSC rescue has been increasingly used for the treatment of several human diseases. However, little is known on the extent of this therapy on the marrow mesenchymal stem cells (MSCs).Aims. To evaluate the feasibility of expansion and multipotencial differentiation of MSCs isolated from patients after HDCT.Patients and Methods. Twelve lymphoma's patients (LP) free of disease in bone marrow (BM) were enrolled in the study. They were submitted to BEAM's protocol with autologous PBSC rescue 28 to 1836 days before the sample collection. Six normal bone marrow donors (ND) were used as controls. The LP and ND median age were 37.5 (range 22–49) and 31.5 years old (range 23–42), respectively. MSCs were isolated by plastic adherence and expanded ex vivo by cultivation in flasks with α-MEM with 15% fetal bovine serum. Media was changed every 3–4 days. At 90% confluence, the cells were re-plated and expanded. The isolation efficiency, colony-forming unit-fibroblast (CFU-F) frequency, growth kinetics, phenotypic characteristics, cell cycle status, multi-lineage differentiation capacity as well as hematopoiesis-supportive function were determined and compared with those of ND-MSCs. This study protocol and the consent form were approved by the institution ethics committees.Results. The results were analyzed by Mann-Whitney test and are expressed as median (range) to LP and ND, respectively. MSCs were successful isolated from all BM samples collected for this study. The cell population showed typical fibroblast-like morphology, appearing as an adherent, spindle shaped cell layer and growing to confluence after a few weeks of culture. The number of CFU-F found at 14 days of culture were 0.94 (0.00–3.75) and 1.25 (0.13–9.25) x10−5 nucleated cells (p = 0.4421). The doubling time between the 1st and 2nd passages was 80.66 (34.08–195.35) and 46.30 (36.36–270.59) hours (p = 0.1025). The cell clones proliferated extensively until 8.17 (1.81–28.27) and 18.11 (11.85–27.48) population doublings (p = 0.0668) in 71.50 (46–88) and 81 (57–103) cultivation days (p = 0.1505). Immunophenotypically, these cells were positive for the CD73, CD105, CD90, CD29, CD13, CD44, CD49e, CD54, HLA-class 1 and Stro-1 markers and negative for CD34, CD45, CD14, CD51/61, HLA-DR and KDR. Regarding the cell cycle status, 85.63 (63.19–92.17) and 82.41 (82.19–87.02) % were in GO-G1 phase (p = 1,000), while only 12.17 (3.33–36.81) and 10.67 (6.59–12.05) % were in S phase (p = 0,6828). All samples tested were capable of differentiating along adipogenic, osteogenic and chondrogenic lineages in vitro, demonstrated by morphology, cyto- and imunohistochemistry or RT-PCR reaction (PPARg and osteopontin genes expression). After co-culture with CD34+ cord blood cells for 1 and 4 weeks, no significant difference CD34+ expansion or colony-forming cells (BFU-E or CFU-GM) were observed between the CD34+ cells/LP-MSCs and CD34+ cells/ND-MSCs co-cultures with cytokines or not.Interpretation and Conclusions. Our results demonstrate that is possible to cultivate and expand MSCs with multipotential differentiation capabilities and hematopoiesis-supportive function from patients after HDCT. Despite there were no significant differences in the median values between LP and ND, the comparative study indicates a possible damage in MSCs by HDCT.

Myeloma Cells Block Runx2/Cbfa1 Activity in Human Bone Marrow Osteoblast Progenitors and Inhibit Osteoblast Formation and Differentiation.

The decreased bone formation contributes to the development of bone lesions in multiple myeloma (MM) patients. Runx2/Cbfa1 is a transcription factor highly restricted to the osteoblastic lineage that has a critical role in the osteoblast formation and differentiation. In this study we investigated the potential effect of myeloma cells on osteoblastogenesis and the role of Runx2/Cbfa1 in the decreased bone formation in MM.In a co-culture system performed in presence or absence of a transwell insert with human myeloma cell lines (HMCLs) (RPMI-8226, U266, XG-1, XG-6, OPM-2) and human bone marrow (BM) pre-osteoblastic cells, obtained from BM stromal cells (BMSC), we observed an inhibition of osteocalcin, alkaline phosphatase and collagen I expression at both mRNA and protein level. Consistently we found that the formation of both Colony Forming Unit Fibroblasts (CFU-F) and Colony Forming Unit Osteoblasts (CFU-OB) were suppressed in long term BM culture by several HMCLs. Moreover, by a gel mobility shift assay (EMSA) we found that Runx-2/Cbfa1 activity was significantly inhibited in BMSC/pre-osteoblastic cells after 48 hours of co-cultures with OPM-2 whereas the level of Runx2/Cbfa1 protein was not affected. The inhibition of osteoblast formation, differentiation and the block of the Runx2/Cbfa1 activity in BMSC/pre-osteoblastic was more pronounced in the cell-to-cell contact conditions as compared to those without the cellular contact.Further we evaluated Runx2/Cbfa1 expression by immunohistochemistry in BM biopsies of 16 MM patients finding a significant reduction of the number of Runx2/Cbfa1 positive cells in osteolytic patients as compared to those without bone lesions (median %: 19.93% vs. 38%; p=0.001) that supports the in vitro results.To identify molecules that could contribute to the inhibitory effect of myeloma cells on osteoblast formation, differentiation and Runx2/Cbfa1 activity, we screened HMCLs and fresh purified CD138+ MM cells for the expression of DKK1, IL-7, noggin, gremlin and secreted frizzled-related protein (sFRP)-2, -3, -4 also evaluating the potential involvement of these osteoblast inhibitors. DKK1 mRNA was expressed by 2 out of 5 HMCLs tested and by 8 out of 12 MM patients tested as well as by BMSC/pre-osteoblastic cells and hOB, however soluble DKK-1 at a widely range of concentration had not effect on the number of CFU-F and CFU-OB in human BM cultures and it did not inhibited Runx2/Cbfa1 activity in BMSC/pre-osteoblastic cells. Moreover, blocking anti-DKK1 Ab did not blunted the effect of HMCLs on osteoblast formation in human BM cultures. All the HMCLs and fresh CD138+ MM cells tested expressed IL-7 mRNA, as we have previously reported. IL-7 reduced Runx2/Cbfa1 activity in BMSC/pre-osteoblastic cells showing an inhibitory effect on CFU-F and CFU-OB formation in BM culture, moreover blocking anti-IL-7 Ab partially reduced the effect of HMCLs in co-cultures. Finally, we found that HMCLs and fresh CD138+ MM cells did not expressed noggin, gremlin, sFRP-2 and rarely produced sFRP-3 and sFRP-4 whereas BMSC/pre-osteoblastic cells and hOB were positive for these factors even if HMCLs had not effect on their expression in co-cultures.In conclusion our data indicate that human myeloma cells inhibit osteoblast formation and differentiation in human BM cultures blocking Runx2/Cbfa1 activity in BMSC/pre-osteoblastic cells through the cell-to-cell contact. Soluble factors produced by myeloma cells as IL-7 could contribute to the inhibitory effect on osteoblastogenesis.

Studies of the route of administration and role of conditioning with radiation on unrelated allogeneic mismatched mesenchymal stem cell engraftment in a nonhuman primate model

Objective The aim of this study was to examine the effects of the route of administration [intrabone marrow (IBM) vs intravenous (IV)] and the role of conditioning with irradiation in optimizing mesenchymal stem cell (MSC) transplantation. Materials and Methods To determine if irradiation resulted in depletion of colony-forming unit fibroblasts (CFU-F), which might favor the engraftment of donor MSC, the number of CFU-Fs was assayed from animals receiving either hemibody irradiation (HBI) or total body irradiation (TBI). Results TBI resulted in a marked reduction of CFU-F numbers that spontaneously resolved, whereas animals receiving HBI did not experience depletion of CFU-F. Animals receiving MSC grafts by the IV route had higher numbers of marrow CFU-F. MSC were transduced using retroviral vectors encoding the neomycin resistance gene (Neo R) and a second gene encoding either the human soluble tumor necrosis factor receptor (hsTNFRII) or β-galactosidase (β-Gal). MSCs were administered by either the IV or IBM route to animals receiving HBI. The Neo R transgene was detectable in hematopoietic tissues of all animals and nonhematopoietic tissues in a single animal. Evidence of transgene expression was documented by detection of β-Gal + cells in BM smears and transiently elevated serum levels of hsTNFRII. Conclusion These studies indicate that 1) MSC possess the ability to engraft and persist in an unrelated mismatched allogeneic hosts; 2) 250-cGy HBI did not favor engraftment of MSC; 3) the IBM route was not more effective than the IV route in delivering MSC grafts; and 4) transplanted MSC preferentially localized to the marrow rather than nonhematopoietic tissues.

Basic fibroblast growth factor corrects proliferative derangement of bone marrow stroma and CD34(+) population following allogeneic stem cell transplantation.

Following stem cell transplantation (SCT), the integrity of the hematopoietic microenvironment is important for the recovery of bone marrow. We studied the effects of basic fibroblast growth factor (bFGF) on the proliferation of clonogenic progenitors and bone marrow stroma from patients receiving cytokine-mobilized allogeneic stem cell transplants (SCT). Patient bone marrow mononuclear cells were studied at a minimum of 6 months after transplantation. Control and patient samples were divided into two fractions, one to establish the adherent stroma layers (SL) and the other to select for the progenitor population. SL from normal subjects or from patients were supplemented with 0 (control), 2, or 20 ng/mL bFGF in combination with heparan sulfate, and the resulting area of the dish covered by stroma was quantitated in each group. At 3 weeks of culture, a monocellular suspension was prepared by exposing SL to 0.1% trypsin. Cells then were cultured in the colony-forming unit fibroblast (CFU-F) assay, which was supplemented with 0 (control), 2, or 20 ng/mL bFGF. With the second fraction, CD34(+) cells were selected with paramagnetic beads, and 1 x 10(4) cells were incubated in cross-culture studies on preformed SL from patient or control origin (with or without the addition of bFGF). SL adherent CD34(+) cells were covered with agar and cultured for 6 days. Aggregates of >20 cells were scored as blastic colonies (CFU-bl). At 3 weeks of culture, the median surface area of dishes covered by monoloayers from 13 patients was 40% (range 10-50% vs normal 55%, range 30-60%; p = 0.00006) and improved significantly, matching control values, in dishes supplemented with bFGF. Addition of bFGF to stroma monolayers had no effect on the number of CFU-F in both patient and control samples. Selected CD34(+) cells from patients receiving transplants and cultured on normal stroma gave significantly fewer CFU-bl than control samples (median 36, range 3-121 vs 147, range 10-184; p = 0.006), but colony numbers corrected following exposure to bFGF. Normal CD34(+) cells proliferated poorly on stroma from patients (median CFU-bl 37.5, range 6-84; p = 0.02), but also expanded significantly following bFGF supplementation (104, range 6-117; p = 0.001). Following SCT, poor SL and CD34(+) proliferation can be corrected by addition of bFGF.

Restoration of murine cytomegalovirus (MCMV) induced myelosuppression by AS101

Infection with cytomegalovirus (CMV) continues to be one of the most common complications following allogeneic bone marrow transplantation. The gravest danger for the host occurs when the virus is reactivated as a result of immunosuppression. In this report we studied the effects of sublethal murine cytomegalovirus (MCMV) infection on the hemopoietic system including bone marrow (BM) cellularity, production of colony stimulating factor (CSF) and interleukin-6 (IL-6) and the development of granulocyte-macrophage colony forming units (CFU-GM), and BM stromal cell viability. Our findings show that the virus infection led to a significant decrease in the number of BM cells and in the production levels of CSF and IL-6. There was also a decrease in the number of stromal cells, as reflected by the number of colony forming unit fibroblasts (CFU-F), and in the relative number of CFU-GM progenitors. Treatment of MCMV infected mice with the immunomodulator AS101 [ammonium trichloro (dioxyethylene 0-0′)tellurate] restored significantly CSF and IL-6 production by BM cells to levels of uninfected control mice as well as the number of CFU-F and stromal cell elements which consequently led to the restoration of the total number of BM cells. Results presented here indicate that AS101 may have immunomodulatory effects on MCMV mediated myelosuppression. Administration of AS101 to patients with CMV associated BM damage may improve the restoration of their BM function.