THE CHARACTERISTICS OF RAT EMBRYONIC FIBROBLASTS (REFS) AND RAT BONE MARROW DERIVED MESENCHYMAL STEM CELLS (RAT-BMMSCS) FROM THE WISTAR RAT: A COMPARATIVE STUDY

Objective: Rat embryonic fibroblasts (REFs) and rat bone marrow-derived mesenchymal stem cells (rat-BMMSCs) an be used as in vitro models for a variety of studies, including for degenerative diseases such as arterial ischemia, tissue engineering and development of induced pluripotent stem cells (iPSCs). Therefore, the further developments of the use of these two cells of great importance. Methods: The experiments were performed with Wistar rat, those with 15-17 d gestation (aged 32 w) as a REFs source and those aged 12 w as a BMMSCs source. Dulbecco's modified eagle medium (DMEM) was used for both cell cultures but with different media supplements. Proliferation ability was determined for both by calculating population doubling time (PDT). Characterization was performed by differentiation testing into osteocyte, chondrocyte and adipocyte cells by staining with Alizarin Red, Alcian Blue and Oil Red O and by an investigation of specific antigen characteristics using flow cytometry with positive CD90 and CD29 and negative CD34 markers. Results: Morphologically, the REFs and rat-BMMSCs had the same fibroblasts like shape. PDT was higher for the REFs than the BMMSCs (p<0.05), and both could differentiate into osteocytes, chondrocytes and adipocyte. The characteristics of the positive markers (CD29 and CD90) were higher in rat-BMMSCs than in REFs. Conclusion: In this study demonstrated that the explant method for REFs isolation and flushing method for rat-BMMSC isolation are both effective. It also showed that rat-BMMSC grow faster than REFs, and that both cells have the same differentiation ability as rat-BMMSCs but with different specific surface antigen characteristics.

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Differential Expression of MicroRNA-3148 in Patients with Osteoporosis and Its Impacts on the Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2021.2635 ◽

2021 ◽

Vol 11 (5) ◽

pp. 957-962

Author(s):

Ainiwaerjiang Damaola ◽

Maerdan Aierken ◽

Mieralimu Muertizha ◽

Abudouaini Abudoureheman ◽

Haishan Lin ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Serum Samples ◽

Alizarin Red ◽

Differentiation Potential ◽

Qrt Pcr ◽

Marrow Mesenchymal Stem Cells ◽

Rat Bone

We aimed to explore the effects of rat bone marrow mesenchymal stem cells (BMSCs) on osteogenic differentiation via analyzing miR-3148 expression in patients with osteoporosis. Realtime quantitative PCR was conducted for assessing microRNA-3148 expression. BMSCs from SD rats were transfected with microRNA-3148 mimics and microRNA-3148 inhibitor via liposomal trans-fection method utilizing Lipo2000, followed by analysis of microRNA-3148 level. After 10-days of osteogenic differentiation induction, alkaline phosphatase (ALP) staining and alizarin red (ARS) staining were done to investigate the osteogenic differentiation potential. Simultaneously, qRT-PCR measured the expression of osteogenesis marker genes (BMP and Runx2) in each group. qRT-PCR analysis revealed a high expression of miR-3148 in the bone tissue and the serum samples from patients with osteoporosis in comparison with healthy individuals. In addition, miRNA-3148 mimics could retard the osteogenic differentiation of BMSCs, while microRNA-3148 inhibitor could prompt the procedure. MicroRNA-3148 was highly expressed in the skeletal tissues and the serum samples from patients with osteoporosis and it could restrain the differentiation of BMSCs into osteoblasts, suggesting that it might be a novel therapeutic target for treating osteoporosis.

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Dihydrotestosterone and 17-Estradiol Enhancement of In Vitro Osteogenic Differentiation of Castrated Male Rat Bone Marrow Mesenchymal Stem Cells (rBMMSCs)

International Journal of Hematology-Oncology and Stem Cell Research ◽

10.18502/ijhoscr.v13i4.1897 ◽

2019 ◽

Author(s):

FAM Abo-Aziza ◽

AA Zaki ◽

AS Amer ◽

RA Lotfy

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Population Doubling ◽

Calcium Deposition ◽

Alp Activity ◽

Marrow Mesenchymal Stem Cells ◽

Rat Bone

Background: In vitro impact of dihydrotestosterone (DHT) and 17-estradiol (E2) in osteogenic differentiation of castrated rat bone marrow mesenchymal stem cells (rBMMSC) still need to be clarified. Materials and Methods: The viability, proliferation and density of cultured rBMMSC isolated from sham operated (Sham) and castrated (Cast) male rats were evaluated. rBMMSC were cultured with osteogenic differentiating medium (ODM) in the presence of DHT (5,10 nM) and E2 (10,100 nM). Osteogenesis was evaluated by alizarin red staining and measurement of calcium deposition and bone alkaline phosphatase (BALP) activity. Results: Population doubling (PD) of rBMMSC isolated from Cast rats was significantly lower (P<0.05) compared to that isolated from Sham rats. rBMMSC from Cast rats showed low scattered calcified nodule after culturing in ODM and did not cause a significant increase in calcium deposition and B-ALP activity compared to rBMMSCs from Sham rats. Exposure of rBMMSC isolated from Cast rats to DHT (5 nM) or E2 (10 nM) in ODM showed medium scattered calcified nodules with significantly higher (P<0.05) calcium deposition and B-ALP activity. Moreover, exposure of rBMMSC to DHT (10 nM) or E2 (100 nM) showed high scattered calcified nodules with higher (P<0.01) calcium deposition and B-ALP activity Conclusion: These results indicated that the presence of testes might participate in controlling the in vitro proliferation and osteogenic differentiation capacity of rBMMSCs. DHT and E2 can enhance the osteogenic capacity of rBMMSCs in a dose-dependent manner. Based on these observations, optimum usage of DHT and E2 can overcome the limitations of MSCs and advance the therapeutic bone regeneration potential in the future.

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308 ISOLATION, DIFFERENTIATION, AND IMMUNOPHENOTYPIC CHARACTERIZATION OF MESENCHYMAL STEM CELLS DERIVED FROM EQUINE ADIPOSE TISSUE AND BONE MARROW

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab308 ◽

2011 ◽

Vol 23 (1) ◽

pp. 250

Author(s):

E. Iacono ◽

B. Merlo ◽

A. Spadari ◽

G. Mari ◽

F. Ricci ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Adipose Tissue ◽

Cellular Therapy ◽

Population Doubling ◽

Blue Staining ◽

Alcian Blue Staining ◽

Alizarin Red ◽

Minimum Criteria

Minimum criteria for the characterisation of human mesenchymal stem cells (MSC) are a) adhesion to the plastic when maintained under culture conditions; b) expression of CD105, CD73, and CD90, and no expression for CD45, CD34, and CD14; and c) differentiation into osteoblasts, adipocytes, and chondroblasts in vitro. One major difficulty in characterising equine MSC is the absence of specific monoclonal antibodies and evidence that certain markers from other species do not cross-react with the equine species. The aim of this work was to isolate, cultivate, differentiate, and conduct cellular characterisation of MSC derived from equine adipose tissue (AT) and bone marrow (BM). Adipose tissue collection was performed at the base of the horses’ tails, and BM was aspirated from the iliac crest. Mononuclear cell fraction was isolated and cultured as previously described by (Colleoni et al. 2009 Vet. Res. Commun. 33, 811–821). Chondrogenic, osteogenic, and adipogenic differentiation were performed in monolayer culture, and evidence for differentiation was made by morphological and cytological evaluations. For molecular characterisation, cells were treated with trypsin, washed with PBS, and fixed with Reagent 1 (Intraprep Kit, Beckman Coulter, Miami, FL, USA), following the manufacturer’s instructions. Samples, after washings, were incubated for 20 min at room temperature with CD105, CD90, CD44, CD45, CD34, CD14, and CD73 mAbs, directly conjugated to fluorescein isothiocyanate, PE, or APC (Beckman Coulter). Appropriate conjugate isotype controls were applied (Beckman Coulter). After staining, cells were washed twice with PBS, and fluorescence intensity was evaluated with a FC500 two-laser equipped cytometer (Beckman Coulter). Results were further analysed with the CXP dedicated program. Samples volumes were 68 ± 23.6 mL for BM and 5.6 ± 1.1 g for AT; in both AT and BM, the isolation rate was 100% (AT: 4/4; BM: 5/5). Undifferentiated cells were passaged up to 8 times for AT and 5 times for BM; population-doubling times (DT) were calculated, and data were analysed by ANOVA (Statistica for Windows, Stat Soft Inc., Tulsa, OK, USA). No significant differences (P > 0.05) were found between DT of all passages. The DT was greater (P < 0.05) for BM (3.2 ± 1.5) than for AT (1.3 ± 0.7). By passage 8, AT MSC underwent 37.3 ± 4.6 cell-doublings (CD); by passage 5, BM MSC underwent 26.2 ± 5.03 CD. Positive von Kossa and Alizarin Red staining confirmed osteogenesis. Alcian blue staining illustrated chondrogenesis, and positive Oil Red O staining suggested adipogenesis. The AT and BM MSC were positive for CD90, CD44, and CD105; all cell lines were negative for haematopoietic markers such as CD34, CD14, and CD45. Although marker CD73 expresses reaction in other studies involving MSC in different species, it did not cross-react with equine AT and BM MSC. Results obtained revealed the immunophenotypic characterisation of the surface of isolated and cultivated MSC, classifying these cells as a promising type of progenitor cells that can be applied in equine cellular therapy.

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