Effects of maternal isocaloric diet containing different amounts of soy oil and extra virgin olive oil on weight, serum glucose, and lipid profile of female mice offspring

Background: Health status of offspring is programmed by maternal diet throughout gestation and lactation. The present study investigates the lasting effects of maternal supplementation with different amounts of soy oil or extra virgin olive oil [EVOO] on weight and biochemical parameters during gestation and lactation of female mice offspring

Methods: Eight weeks old female C57BL/6 mice [n=40] were assigned through simple randomization into four isocaloric dietary groups [16% of calories as soy oil [LSO] or EVOO [LOO] and 45% of calories as soy oil [HSO] or EVOO [HOO]] during three weeks of gestation and lactation. After weaning [at 3 weeks], all offspring received a diet containing 16% of calories as soy oil and were sacrificed at 6 weeks. Two-way ANOVA was used to adjust for confounding variables and repeated measures test for weight gain trend. Statistical analyses were performed with the IBM SPSS package

Results: At birth and adolescence, the weight of offspring was significantly higher in the soy oil than the olive oil groups [P<0.001 and P<0.001, respectively]. Adolescence weight was significantly higher in the offspring born to mothers fed with 16% oil than those with 45% oil [P=0.001]. Serum glucose, triglyceride and total cholesterol were significantly higher in the LSO than LOO [P<0.001, P<0.001 and P<0.001], LSO than HSO [P<0.001, P=0.03 and P<0.001], and LOO than HOO [P<0.001, P<0.001 and P<0.001] dietary groups, respectively. Serum triglyceride and total cholesterol were significantly higher in the offspring of HSO than HOO fed mothers [P<0.001 and P<0.001, respectively]

Conclusion: A maternal diet containing EVOO has better effects on birth weight, as well as weight and serum biochemical parameters in offspring at adolescence

Effects of exercise prior or during pregnancy in high fat diet fed mice alter bone gene expression of female offspring: an experimental study

Background: Based on different studies it was shown that exercise training is an important factor in preconception and prenatal care

Objective: The aim of this study was to determine whether regular preconception exercise training with or without exercise training during pregnancy decreases detrimental effects of maternal high fat diet on female offspring bone health

Materials and Methods: Twenty-four C57BL/6 female mice were fed high-fat diet [35%] and were randomly divided into four groups: trained in preconception period and exercised during pregnancy [TE]; trained in preconception periods but unexercised during pregnancy [TC]; untrained in preconception periods but exercised during pregnancy [CE]; untrained and unexercised [CC]. Trained mice were subjected to a protocol of moderate endurance exercise training over a period of 4 weeks before pregnancy. TE and CE Dams groups had access to wheels throughout pregnancy until delivery. Analyses were performed on the female offspring that did not have access to running wheels or exercise training during any portion of their lives. The relative expression levels of beta-catenin, Peroxisome proliferator-activated receptor Y [PPARY], osteoprotegerin [OPG], and Receptor activator of nuclear factor NF-kB ligand [RANKL] were determined by Quantitative RT-PCR [qPCR]

Results: Exercise during pregnancy in isolation had no effect on any measure genes but exercise both before and during pregnancy affected all genes. Exercise only before pregnancy increased beta-catenin and OPG and decreased PPARY, RANKL, and RANKL/OPG ratio [p<0.001]

Conclusion: This study demonstrated that maternal exercise training before and during pregnancy may modulate the risk of bone disorders in offspring of mothers fed a high-fat diet

Mesenchymal stromal cells implantation in combination with platelet lysate product is safe for reconstruction of human long bone nonunion

Objective: nonunion is defined as a minimum of 9 months since injury without any visible progressive signs of healing for 3 months. Recent literature has shown that the application of mesenchymal stromal cells is safe, in vitro and in vivo, for treating long bone nonunion. The present study was performed to investigate the safety of mesenchymal stromal cell [MSC] implantation in combination with platelet lysate [PL] product for treating human long bone nonunion

Materials and Methods: in this case series clinical trial, orthopedic surgeons visited eighteen patients with long bone nonunion, of whom 7 complied with the eligibility criteria. These patients received mesenchymal stromal cells [20 million cells implanted once into the nonunion site using a fluoroscopic guide] in combination with PL product. For evaluation of the effects of this intervention all the patients were followed up by taking anterior-posterior and lateral X-rays of the affected limb before and 1, 3, 6, and 12 months after the implantation. All side effects [local or systemic, serious or non-serious, related or unrelated] were observed during this time period

Results: from a safety perspective the MSC implantation in combination with PL was very well tolerated during the 12 months of the trial. Four patients were healed; based on the control X- ray evidence, bony union had occurred

Conclusion: results from the present study suggest that the implantation of bone marrow-derived MSCs in combination with PL is safe for the treatment of nonunion. A double blind, controlled clinical trial is required to assess the efficacy of this treatment [Registration Number: NCT01206179]

Transplantation of Autologous Bone Marrow Mesenchymal Stem Cells into the Testes of Infertile Male Rats and New Germ Cell Formation

BACKGROUND: Mesenchymal stem cells (MSCs), have been suggested as a potential choice for treatment of male infertility. Yet, the effects of MSCs on regeneration of germinal epithelium of seminiferous tubules and recovery of spermatogenesis have remained controversial. In this research, we have evaluated and compared the fate of autologous bone marrow (BM)-MSCs during three different periods of time- 4, 6 and 8 weeks after transplantation into the testes of busulfan-induced infertile male rats. METHODS: Rats BM samples were collected from tibia bone under anesthesia. The samples were directly cultured in culture medium. Isolated, characterized and purified BM-MSCs were labeled with PKH26, and transplanted into the testes of infertile rats. After 4, 6 and 8 weeks, the testes were removed and underwent histological evaluations. RESULTS: Immunohistochemical analysis showed that transplanted BM-MSCs survived in all three groups. Some of the cells homed at the germinal epithelium and expressed spermatogonia markers (Dazl and Stella). The number of homed spermatogonia-like cells in 4-week testes, was more than the 6-week testes. The 8-week testes had the least numbers of homed cells (p<0.05). Immunostaining for vimentin showed that BM-MSCs did not differentiate into the sertoli cells in the testes. CONCLUSIONS: From our results, it could be concluded that, autologous BM-MSCs could survive in the testis, migrate onto the seminiferous tubules basement membrane and differentiate into spermatogonia. Although, no more differentiation was observed in the produced spermatogonia, generation of such endogenous GCs would be a really promising achievement for treatment of male infertility using autologous stem cells.

In vitro differentiation of human umbilical cord blood CD133[+] cells into insulin producing cells in co-culture with rat pancreatic mesenchymal stem cells

Pancreatic stroma plays an important role in the induction of pancreatic cells by the use of close range signaling. In this respect, we presume that pancreatic mesenchymal cells [PMCs] as a fundamental factor of the stromal niche may have an effective role in differentiation of umbilical cord blood cluster of differentiation 133[+] [UCB-CD133[+]] cells into newly-formed beta-cells in vitro. This study is an experimental research. The UCB-CD133[+] cells were purified by magnetic activated cell sorting [MACS] and differentiated into insulin producing cells [IPCs] in co-culture, both directly and indirectly with rat PMCs. Immunocytochemistry and enzyme linked immune sorbent assay [ELISA] were used to determine expression and production of insulin and C-peptide at the protein level. Our results demonstrated that UCB-CD133[+] differentiated into IPCs. Cells in islet-like clusters with [out] co-cultured with rat pancreatic stromal cells produced insulin and C-peptide and released them into the culture medium at the end of the induction protocol. However they did not respond well to glucose challenges. Rat PMCs possibly affect differentiation of UCB-CD133[+] cells into IPCs by increasing the number of immature beta-cells

Small molecule-BIO accelerates and enhances marrow-derived mesenchymal stem cell in vitro chondrogenesis

Background: Hyaline cartilage defects exhibit a major challenge in the field of orthopedic surgery owing to its limited repair capacity. On the other hand, mesenchymal stem cells [MSCs] are regarded as potent cells with a property of cartilage regeneration. We aimed to optimize marrow-derived MSC chondrogenic culture using a small bioactive molecule referred to as BIO

Methods: MSCs from the marrow of NMRI mice were extracted, culture-expanded, and characterized. Micro-mass culture was then established for chondrogenic differentiation [control group]. The cultures of MSC in chondrogenic medium supplemented with 0.01, 0.05, 0.1, and 1 microM BIO were taken as the experimental groups. Cartilage differentiation was examined by both histological sections and real-time PCR for Sox9, aggrecan, and collagen II at different time points. Moreover, the involvement of the Wnt pathway was investigated

Results: Based on histological sections, there was seemingly more intense metachromatic matrix produced in the cultures with 0.01 microM BIO. In this experimental group, cartilage-specific genes tended to be upregulated at day 14 compared to day 21 of the control group, indicating the accelerating effect of BIO on cartilage differentiation. Overall, there was statistically a significant increase [P=0.01] in the expression level of cartilagespecific genes in cultures with 0.01 microM BIO [enhancing effects]. These upregulations appeared to be mediated through the Wnt pathway evident from the significant upregulation of T-cell factor and beta-catenin molecules [P=0.01]

Conclusion: Taken together, BIO at 0.01 microM could accelerate and enhance in vitro chondrogenesis of mouse marrow-derived MSCs

Epigenetic regulation of osteogenic and chondrogenic differentiation of mesenchymal stem cells in culture

Management of mesenchymal stem cells [MSCs] capabilities to differentiate in to osteogenic and Chondrogenic lineages would be of utmost importance for their future use in difficult to treat cases of destroyed bone and cartilage. Thus, an understanding of the epigenetic mechanisms as important modulators of stem cell differentiation might be useful. Epigenetic mechanism refers to a process that regulates heritable and long-lasting alterations in gene expression without changing the DNA sequence. Such stable changes would be mediated by several mechanisms including DNA methylation and histone modifications. The involvement of epigenetic mechanisms during MSC bone and cartilage differentiation has been investigated during the past decade. The purpose of this review is to cover outstanding research works that have attempted to ascertain the underlying epigenetic changes of the nuclear genome during in vitro differentiation of MSCs into bone and cartilage cell lineages. Understanding such genomic alterations may assist scientists to develop and recognize reagents that are able to efficiently promote this cellular differentiation. Before summarizing the progress on epigenetic regulation of MSC bone and cartilage differentiation, a brief description will be given regarding in vitro conditions that favor MSC osteocytic and chondrocytic differentiation and the main mechanisms responsible for epigenetic regulation of differentiation

Intra-articular injection of autologous mesenchymal stem cells in six patients with knee osteoarthritis

Osteoarthritis [OA] is a progressive disorder of the joints caused by gradual loss of articular cartilage, which naturally possesses a limited regenerative capacity. In the present study, the potential of intra-articular injection of mesenchymal stem cells [MSCs] has been evaluated in six osteoarthritic patients. Six female volunteers, average age of 54.56 years, with radiologic evidence of knee OA that required joint replacement surgery were selected for this study. About 50 ml bone marrow was aspirated from each patient and taken to the cell laboratory, where MSCs were isolated and characterized in terms of some surface markers. About 20-24x106 passaged-2 cells were prepared and tested for microbial contamination prior to intra-articular injection. During a one-year follow-up period, we found no local or systemic adverse events. All patients were partly satisfied with the results of the study. Pain, functional status of the knee, and walking distance tended to be improved up to six months post-injection, after which pain appeared to be slightly increased and patients' walking abilities slightly decreased. Comparison of magnetic resonance images [MRI] at baseline and six months post-stem cell injection displayed an increase in cartilage thickness, extension of the repair tissue over the subchondral bone and a considerable decrease in the size of edematous subchondral patches in three out of six patients. The results indicated satisfactory effects of intra-articular injection of MSCs in patients with knee OA

Amniotic fluid stem cells and their application in cell-based tissue regeneration

Advances in stem cell biotechnology hold great promise in the field of tissue engineering and regenerative medicine. Of interest are marrow mesenchymal stem cells [MSCs], embryonic stem cells [ESCs], and induced pluripotent stem cells [iPSCs]. In addition, amniotic fluid stem cells [AFSCs] have attracted attention as a viable choice following the search for an alternative stem cell source. Investigators are interested in these cells because they come from the amniotic fluid that is routinely discarded after birth. There have been multiple investigations conducted worldwide in an attempt to better understand AF-SCs in terms of their potential use in regenerative medicine. In this review we give a brief introduction of amniotic fluid followed by a description of the cells present within this fluid. Their history related to stem cell discovery in the amniotic fluid as well as the main characteristics of AF-SCs are discussed. Finally, we elaborate on the potential for these cells to promote regeneration of various tissue defects, including fetal tissue, the nervous system, heart, lungs, kidneys, bones, and cartilage.

Enhancement of glycosaminoglycan-rich matrix production in human marrow-derived mesenchymal stem cell chondrogenic culture by lithium chloride and SB216763 treatment

Cartilage mass produced from mesenchymal stem cell [MSC] differentiation would be a suitable candidate for use in regenerative medicine. Since the proper function of cartilage tissue is largely dependent on matrix glycosaminoglycan [GAG] contents, the objective of this study was to investigate the enhancing effect of two GSK3 inhibitors on the GAG content of cartilage produced by human marrow MSCs in vitro chondrogenesis. MSCs that were used in this experimental study were derived from human marrow aspirates and confirmed using standard assays. Optimal concentrations of Lithium chloride and SB216763 were determined based on the yield of viable cell numbers in MSC cultures treated with varying concentrations of either Lithium chloride or SB216763.Passaged-3 MSCs were then centrifuged into small aggregates and provided with a chondrogenic medium supplemented with either lithium or SB216763 reagent at the optimal concentration determined in the previous experiment. Three weeks after, GAG contents of the culture were quantified and compared to each other and the control. According to our data, the cultures treated with 5 mM Lithium and 1 micro M SB216763 tended to have comparatively more viable cells; therefore these concentrations were used in the differentiation experiments. The addition of either SB216763 or lithium to chondrogenic cultures appeared to significantly enhance cartilage matrix production. In SB216763 and Lithium-treated cultures average GAG concentrations were 6.17 +/- 0.7 and 6.12 +/- 1.1 micro g/ml compared to 2.00 +/- 0.3 micro g/ml in the control [p<0.05]. Using SB216763 and Lithium as supplements in human marrow MSC chondrogenic culture can lead to the production of cartilage mass high in GAG content

Mesenchymal stem cells from murine amniotic fluid as a model for preclinical investigation

Despite the suitability of a mouse model for preclinical investigations; little is known regarding mesenchymal stem cells derived from murine amniotic fluid. This is the subject of the present study. Amniotic fluid was collected from NMRI mice during the second weeks of pregnancy and plated. The cells that adhered to the culture surfaces were propagated with three successive subcultures and then characterized. To determine the differentiation potential, the cells were cultivated under osteogenic, adipogenic, and chondrogenic conditions, and followed by specific staining and RT-PCR analysis for differentiation. The proliferative potential of the cells were with clonogenic assays, population doubling time and number and by growth curve plotting. Cellular aging was investigated with the senescence-associated beta-galactosidase staining method. The amniotic fluid primary cell culture was composed of round flattened and fibroblastic cells. The latter dominated the culture after several passages. Successful tripotent differentiation of the isolated cells into bone, cartilage and adipose cells were indicative of their mesenchymal stem cells nature. The isolated cells appeared to be relatively proliferative cells as confirmed by the population doubling time value which was equal to about 69 hours. Furthermore, the cells were relatively clonogenic and they tended to initate proliferation immediately after plating [there was no lag phase in their growth curve]. Beta- galactosidase positive cells were first observed at passage 3 and increased in number with subsequent passagers. Collectively it was concluded that murine amniotic fluid contained mesenchymal stem cells with relatively high proliferation property and typical tripotent differentiation potential

Mesenchymal stem cells derived from rat epicardial versus epididymal adipose tissue

Some investigation has indicated that adipose-derived stem cells possess different surface epitopes and differentiation potential according to the localization of fat pad from which the cells were derived. In the present study proliferation capacity and aging of such cells were explored. Adherent cells were isolated from the collagenase digests of adipose tissues excised from rat epicardial and epididymal regions and propagated with several subcultures. The cells were then investigated whether or not they were able to differentiate into bone, cartilage and adipose cell lineages. Studied cells from two adipose tissues were also compared with respect to their in vitro proliferation capacity. The presence of senescent cells in the culture was determined and compared using senescence-associated [SA] beta-galactosidase staining method. Successful differentiations of the cells were indicative of their mesenchymal stem cells [MSCs] identity. Epicardial adipose-derived cells tended to have a short population doubling time [45 +/- 9.6 hr] than the epididymal adipose-derived stem cells [69 +/- 16 hr, P< 0.05]. Colonogenic activity and the growth curve characteristics were all better in the culture of stem cells derived from epicardial compared to epididymal adipose tissue. Comparatively more percentage of senescent cells was present at the cultures derived from epididymal adipose tissue [P< 0.05]. Our data emphasize on the differences existed between the stem cells derived from adipose depots of different anatomical sites in terms of their proliferative capacity and in vitro aging. Such data can help understand varying results reported by different laboratories involved in adipose stem cell investigations

Study of mesenchymal stem cell proliferation and bone differentiation on composite scaffolds of PLLA and nano hydroxyapatite with different morphologies

Nowadays, bone constructs elaborated according to tissue engineering principles are being regarded as an ideal choice for the reconstruction of segmental bone defects. In this study, proliferation and bone differentiation of marrow-derived mesenchymal stem cells [MSCs] were compared in different composite scaffolds containing varying morphologies of nano hydroxyapatite [nHAP]. Needle nHAP/PLLA [poly [L-lactide acid]], spherical nHAP/ PLLA and rod nHAP/PLLA scaffolds were prepared and 3D cultures of passaged-3 rat MSCs were established using the scaffolds. The loading of the cells onto the scaffold internal spaces was confirmed with microscopy and their proliferation was determined by MTT assay. To compare the osteogenic differentiation of the cells on the scaffold surfaces, osteogenic 3D cultures were established and kept for 21 days. At the end of this period culture mineralization and relative bone-related gene expression were quantified using the alizarin red quantification assay and semi quantitative RT-PCR analysis respectively. ANOVA was used to compare the data. According to the MTT assays, cells adhered to all the studied scaffold surfaces tended to proliferate. In this respect the microenvironment provided by the needle nHAP/ PLLA appeared much better than that of either the spherical or rod nHAP/PLLA scaffolds [P<0.05]. Similarly, mineralization was observed to be heavier for the needle nHAP/PLLA scaffold compared to the two other composite scaffolds. In addition, the relative expression of coll I, osteocalcin, runx2 and ALP genes all appeared to be significantly higher in the cells cultivated on needle nHAP/PLLA scaffolds versus their spherical and rod counterparts. Overall, needle nHAP/PLLA scaffolds appear to provide the most appropriate matrix for producing bone construct using MSCs

[Comparison of proliferation and osteoblast differentiation of marrow-derived mesenchymal stem cells on nano- and micro-hydroxyapatite contained composite scaffolds]

Bones constructed by tissue engineering are being considered as valuable materials to be used for regeneration of large defects in natural bone. In an attempt to prepare a new bone construct, in this study, proliferation and bone differentiation of marrow-derived mesenchymal stem cells [MSCs] on our recently developed composite scaffolds of nano-, micro-hydroxyapatite/poly[l-lactic acid] were compared with pure poly[l-lactic acid] scaffolds. For this purpose, some passaged-3 rat MSCs were three-dimensionally cultivated on the scaffold surfaces and their morphology was observed with scanning electron microscopy. Cell proliferations on different scaffolds were examined by MTT assays. Osteogenic cultures were established with the scaffolds loaded with MSCs for 21 days at the end of which culture mineralization; the cell alkaline phosphatase [ALP] Level and the relative expression of selected bone specific genes were quantified and compared to each other. Our results indicated that the cells having been adhered and assumed spherical morphology were able to proliferate in all studied scaffolds. The microenvironment provided by nano-scaffolds appeared much better medium than those of micro-scaffolds and pure PLLA [P < 0.05]. The osteogenesis assays indicated to the superiority of nanoscaffolds in supporting MSCs undergoing bone differentiation, which was reflected in high cellular ALP levels, increased bone-related gene expression and enhanced culture mineralization. Collectively, the bone construct prepared with nano-hydroxyapatite/ poly [llactic acid] scaffold and proliferated MSCs would be suitable candidate for use in bone regenerative medicine

Isolation and in vitro characterization of mesenchymal stem cells derived from the pulp tissue of human third molar tooth

It is still controversial that the stem cells isolated from human dental pulp meets the criteria for mesenchymal stem cells [MSCs]. The aim of the present study was to examine whether or not they are MSCs, or are distinct stem cells population residing in tooth pulp. Adherent fibroblastic cells in the culture of pulp tissue from human third molars were propagated through several successive subcultures. Passaged-3 cells with a tendency to differentiate into odontoblastic cells were used to examine the key properties of MSCs including typical tripotent differentiation potential into bone, cartilage and adipose cell lineages and the expression of typical surface antigens. Moreover, they were examined for growth capacity in culture. Dental pulp stem cells successfully progressed towards differentiation among three skeletal cell lineages. More than 90% of the cell population exhibited the expression of surface antigens known to be found on mesenchymal lineages such as CD105, CD90, CD44, and CD73, while only less than 2% expressed endothelial-hematopoietic epitopes including CD56, CDllb, CD34, CD31, CD33, and CD45. The cells exhibited a relatively high proliferation capacity with population doubling time of about 21.9 hours. The dental pulp stem cells are of MSC population, and may be considered suitable for use in regenerative medicine, owing to their relatively rapid rate of in vitro propagation

Matrigel enhances in vitro bone differentiation of human marrow-derived mesenchymal stem cells

The use of co-culture cells as well as extra cellular matrix are among those strategies that have been employed to direct mesenchymal stem cell [MSC] bone differentiation in culture. In this regard, there is no study considering the effects of Matrigel on mesenchymal stem cell [MSC] in vitro bone differentiation. This was the subject of the present study. Human passaged-3 MSCs isolated from the marrow aspirates were seeded on either Matrigel or conventional polystyrene plastic surfaces [as control] for 10 days. To compare the cell proliferation in two cultures, the cell numbers were determined during the cultivation period. For bone differentiation, the confluent cultures from either group were provided with osteogenic medium and incubated for 21 days during which the alkaline phosphates [ALP] activity, culture mineralization and the expression of some bone-related genes were quantified and statistically compared. MTT assay indicated that Matrigel-cultivated cells underwent statistically less proliferation than polystyrene-cultivated cells [P<0.05]. Regarding the osteogenic differentiation, ALP activity was significantly high in Matrigel versus plastic cultures. Calcium deposition in Matrigel cultures tended to be significantly extensive compared with that of control cultures [2.533 +/- 0.017 versus 0.607 +/- 0.09 mM]. Furthermore, according to the semi-quantitative RT-PCR analysis, compared with polystyrene plastic surface, Matrigel seemed to provide a microenvironment in which human MSC expressed osteocalcin and collagen I genes in a significantly higher level. Collectively it seems that Matrigel could be considered as an appropriate matrix for MSC osteogenic differentiation

[Quantitative expression of BAG1, BAX and BCL-2 genes in human embryos with different fragmentation grades derived from ART]

The purpose of this study was to evaluate the quantitative expressions of BAG1, BAX and BCL-2 in human embryos with different fragmentation grades as derived from assisted reproduction technology [ART]. Fragmented and normal human 8-cell embryos were scored according to the degree of fragmentation with an inverted microscope and divided into four grades [grade I: no or minimal fragmentation [<5%], grade II: embryos with <25% fragmentation, grade III: embryos with >25% fragmentation and grade IV: apoptotic induced embryos with actinomycin D]. In this study, TUNEL labeling was initially used to detect apoptosis, and then revers transcription polymerase chain reaction [RT-PCR] and quantitative PCR were used to define the quantitative expressions of experimental genes in human embryos with different fragmentation grades. The results of TUNEL labeling showed that embryos with higher fragmentation had a high number of apoptotic bodies. The results of RT-PCR and q-PCR analyses showed a significantly decreased amount of BAGI transcript expression from group I to group IV. The highest expression of BAX gene was observed in group II, however, the transcript of BCL-2 gene was not observed in any of the experimental groups. The effect of actinomycin D on transcript expression amounts of experimental genes in apoptotic induced embryos [group IV] compared to control embryos [group I] showed a significant decrease. mRNA expression of BAG1 gene can be used as a good marker to detect apoptosis in human embryos. However, the transcript of BCL-2 gene does not play a role in the detection of apoptosis in human embryos at the 8-cell stage

Ex vivo expansion and differentiation of mesenchymal stem cells from goat bone marrow

Mesenchymal stem cells [MSCs] from large animals as goat which is genetically more closely related to human have rarely been gained attentions. The present study tried to isolate and characterize MSCs from goat bone marrow. Fibroblastic cells appeared in goat marrow cell culture were expanded through several subcultures. Passaged-3 cells were then differentiated among the osteogenic, adipogenic and chondrogenic cell lineages to determine their MSC nature. Differentiations were determined by RT-PCR analysis of related gene expression. To identify the best culture conditions for propagation, passage-3 cells were plated either at varying cell densities or different fetal bovine serum [FBS] concentrations for a week, at the end of which the cultures were statistically compared with respect to the cell proliferation. In this study, we also determined goat MSC population doubling time [PDT] as the index of their in vitro expansion rate. Passage-3 fibroblastic cells tended to differentiate into skeletal cell lineages. This was evident in both specific staining as well as the specific gene expression profile. Moreover, there appeared to be more expansion when the cultures were initiated at 100 cells/cm[2] in a medium supplemented with 15% FBS. A relatively short PDT [24.94 +/- 2.67 hr] was a reflection of the goat MSC rapid rate of expansion. Taken together, fibroblastic cells developed at goat marrow cell culture are able to differentiate into skeletal cell lineages. They undergo extensive proliferation when being plated at low cell density in 15% FBS concentration

Bio treatment protects rat marrow-derived mesenchymal stem cell culture against the TNF-a induced apoptosis

This study is an attempt to examine the anti apoptotic effects of BIO on rat MSC culture. Rat marrow primary cell culture was established and exposure groups were defined; cultures with 0.01, 0.1, 1 micro M BIO. Cells cultured without BIO treatment were used as controls. During culture expantion, the average doubling time, as an index of the rate of cell growth, were determined and compared. To examine whether or not BIO is able to protect MSCs against apoptosis, the passaged-3 cells from each group were induced to undergo apoptosis with the addition of TNF-alpha [Tumor necrotic factor-alpha]. Three days after, the cultures were quantified in terms of the percentages of apoptotic cells using either the Tunnel or Annexin V staining method. Marrow cells cultivated with 0.1 and 1 micro M BIO appeared to expand at a significantly more rapid rate than the 0.01 micro M BIO and the control cultures [p < 0.05]. Tunnel staining indicated that in 1 micro M BIO-treated groups, there were lower percentages of apoptotic nuclei than in groups with other concentrations of BIO [p < 0.05]. The BIO protective effect appeared to be dose-dependent in that the cultures with high BIO content possessed less apoptotic nuclei. The results obtained by Annexin staining were in agreement with the results of Tunnel staining. The Annexin method additionally takes into account the early apoptotic cells which are not detectable by the Tunnel method. Taken together, it seems that cultivation with BIO could both increase the growth rate of marrow cells and protect MSCs against induced apoptosis

Quantitative analysis of the proliferation and differentiation of rat articular chondrocytes in alginate 3D culture

While articular chondrocytes are among those appropriate candidates for cartilage regeneration, the cell dedifferentiation during monolayer culture has limited their application. Several investigations have indicated the usefulness of alginate, but the topic of proliferation and differentiation of chondrocytes in alginate culture has still remained controversial. Rat articular chondrocytes were released by enzymatic digestion, plated at 5 x 10[4] cells/cm[2] and culture-expanded. Passaged-5 cells were then cultivated in alginate as 2-mm beads for a period of two months during which the expansion rate and the level of cell differentiation were determined by [3-[A, 5- dimethylthiazolyl- 2-yl]-l, 5-diphenyl tetrazolium bromide] assay and real-time PCR analysis respectively and compared with those of chondrocytes in monolayer culture. Average population doubling time in alginate cultures [10.04 +/- 0.9 days] tended to be significantly [P<0.05] higher than that in monolayer cultures [2.94 +/- 0.3 days]. The period of alginate culture could be subdivided into expansion phase [Days 0-40]; during which proliferation appeared to be high and differentiation phase [Days 40-60] during which the expression of cartilage-specific genes including collagen II and aggrecan tended to be upregulated. During both the proliferation and differentiation phases, the expression of collagen I was low. At chondrocytes monolayer cultures, the proliferating cells appeared to have a very low expression level of cartilage-specific genes and a high expression level of collagen I gene during the entire culture period [P<0.05]. It seems that alginate provides conditions in which rat articular chondrocytes are able to undergo proliferation and differentiation in certain time point of cultivation period