Study on the biological characteristics of rat bone marrow mesenchymal stem cells

AIM: To investigate the basic biological characteristics of adult rat bone marrow mesenchymal stem cells(rBMMSCs), and compare to that of human BMMSCs (hBMMSCs). METHODS: rBMMSC and hBMMSCs were separated from bone marrow with the difference of adherence and Ficoll-Paque reagent, and expanded in culture medium in vitro, respectively. The proliferation and growth characteristics of the primary and different passage culture of rBMMSCs and hBMMSCs were analysed. The neural differentiation capacity of rBMMSCs with passages were observed. To detect the surface antigens of rBMMSCs, the labeled cells were analysed on a FACScan flow cytometer. The karyotype of rBMMSCs were detected by blocking cellular fission with colchicines. RESULTS: rBMMSCs and hBMMSCs have a strong self-renewal capacity. Approximately (4-8)×10¹² and (3-4)×10¹² cells were obtained after passage 15 in vitro, respectively. The ability of proliferation, CFU-Fs, and neural differentiation of rBMMSCs and hBMMSCs were decreased gradually with passages, but the ability of proliferation and CFU-Fs of rBMMSCs were higher than that of hBMMSCs at different passage. FACScan result showed rBMMSCs were uniformly positive for CD29 and CD44, and negative for CD11b, CD45, CD61, CD71, CD80, CD86,MHCⅠ and MHCⅡ. rBMMSCs had an normal karyotype, which had an average of 37.0±4.0 to 40.5±2.5 chromosomes. CONCLUSION: Adult rBMMSCs have strong self-renewal and neural differentiation capacity, and have an normal karyotype. So rBMMSCs can be used as the seed cells for tissue engineering.     

Expression of PCNA,IL-6,IL-11 and galectin-3 in human umbilical cord mesenchymal stem cells during long-term culture

AIM: To investigate the changes of the mRNA and protein levels of proliferating cell nuclear antigen(PCNA),interleukin-6(IL-6),IL-11 and galectin-3 in human umbilical cord mesenchymal stem cells (hUC-MSCs) during long-term culture in vitro. METHODS: Human umbilical cords from neonates via caesarean section were collected, and the hUC-MSCs were isolated and sub-cultured. The cells and culture supernatants at passages 3, 8, 18, 28 and 33 were collected. The mRNA expression and protein secretion of PCNA,IL-6,IL-11 and galectin-3 in different passages of hUC-MSCs were detected by qRT-PCR, ELISA, and Western blotting. RESULTS: The mRNA expression of PCNA,IL-6 and IL-11 and protein secretion of IL-6 and IL-11 reduced gradually along with the passage.Comparing the 33rd passage with the 3rd passage of MSCs, the mRNA expression of PCNA,IL-6 and IL-11 decreased by 33%, 56% and 37%, and the protein secretion decreased by 50.3% and 58.9%, respectively. The differences were statistically significant (P<0.01). No statistically significant difference of galectin-3 mRNA expression and the protein band brightness (P>0.05) in the MSCs among different passages was observed. CONCLUSION: The multiplication and haematogenesis support capabilities of the might decrease or even lose during long-term culture in vitro. Long-term culture might have no effect on hUC-MSCs immunoregulatory capability of hUC-MSCs, which requires further study.     

Osteogenic and neurogenic differentiation of human yolk sac mesenchymal stem cells

AIM: To purify human yolk sac mesenchymal stem cells (hYS-MSC) and investigate its osteogenic and neurogenic differentiation potentials. METHODS: hYS-MSC were separated from yolk sac and purified via passage culture. The karyotype of hYS-MSCs was analyzed via G-banded characteristics. Flow cytometric analysis was used to determine the cell cycle and phenotype of hYS-MSC. The AKP expression of hYS-MSC was also tested. Osteogenic differentiation of hYS-MSCs was induced by 10-8mol/L dexamethasone, 10 mmol/L β-glycerophosphate and 50 mg/L vitamin C. Alizarin red S stain was used for identification of mineralization. β-mecaptoethanol or salviae miltiorrhizae were used to induce neurogenic differentiation of hYS-MSCs. The expressions of NSE, NF and GFAP were identified by immunohistochemical method. RESULTS: hYS-MSCs could be purified at passages 2 or 3. The cell cycle analysis suggested that hYS-MSCs showed strong proliferational potentials by which the cells kept normal diploid karyotype during the in vitro culture. Flow cytometry showed the phenotype of purified hYS-MSCs was uniformly positive for CD29, CD44, CD105, and CD166, and negative for reactivity to antigens CD34, CD45, or CD86. hYS-MSCs were weakly but clearly positive in AKP. Osteogenic differentiation was appeared after induction of osteogenic differentiation. hYS-MSCs, which were of spindle shape, uniform in size, were induced to pleomorphism osteoblast-like cells which expressed high level of AKP. Aggregates or nodules were formed at day 7 and calcium accumulation was detected by alizarin red S staining on day 10 or day 14. Neurogenic differentiation of hYS-MSCs was induced by β-mecaptoethanol or salviae miltiorrhizae. NSE, NF or GFAP positive cells were detected by immunohistochemical staining. CONCLUSIONS: hYS-MSCs have strong proliferation potential and the normal diploid karyotype is kept during the in vitro culture. The phenotype of hYS-MSCs is coincident with adult hMSCs. hYS-MSCs could be induced to differentiate into osteogenic or neurogenic cells.     

Gene transfer into different passages of bone marrow mesenchymal stem cells

AIM: To investigate the efficiency and stability of adenovirus-medicated gene transfer into different passages of bone marrow mesenchymal stem cells (BMSCs). METHODS: BMSCs were obtained from bone marrow of SD rats and cultured. Then passage 3 (P3) and P8 BMSCs were transfected with Ad-CMV-GFP, respectively. The transfection ratio was evaluated by flow cytometry. At the same time coxsackie and Ad receptor (CAR) of different passages of BMSCs was estimated by RT-PCR and Western blotting. RESULTS: The green fluorescence was observed 24 h after transfection, while the strength of fluorescence increased with time and the peak was at 7 days. It was seen that the transfection ratio was over 80% and there was no difference between P3 and P8 BMSCs (P>0.05). Flow cytometry analysis by different gates showed the transfection ratio was high in BMSCs in the period of productive metabolism. The mRNA expression of CAR in P3, P6 and P8 was similar, and the same change was observed in the protein expression of CAR in P3 and P8 BMSCs. CONCLUSION: Ad-CMV-GFP is transferred to BMSC effectively and sustained about 28 days. It is suspected that BMSCs in mitotic phase are easy to be transferred by Ad-CMV-GFP and different passages of BMSCs from P3 to P8 BMSCs can be as high-effectively gene vehicle.     

Alteration of apoptotic susceptibility and bcl-2 gene in malignant transformation of human bronchial epithelial cells

AIM: To demonstrate the susceptibility of cell apoptosis varies during the progress of cell malignant transformation from human being in vitro. METHODS: A SV₄₀T-transfected human bronchial epithelial immortalized cell line (called M) was selected in this work, which has acquired some characteristics of malignant transformation at the later passage. The alterations of apoptosis and bcl-2, P53 genes between early and later passage of M cells were investigated by means of TDT labeling in situ, chromosome FISH, RNA and protein testing, etc. RESULTS: Incidence of apoptosis induced by cis-platin was significantly lower in later than in early passages of M. Levels of bcl-2 mRNA and protein in later passages were higher than early passages of M, and overexpression of bcl-2 was accumulated following the development of cellular malignancy. P53 protein level was as high in early as in later passages. CONCLUSION: Overexpression of bcl-2 decreases the cellular sensitivity to apoptotic inductors plays an important role during progress of carcinogenesis in human bronchial epithelial cancers. The inactivation of P53 protein in the SV₄₀-T transfected M cell line may be one of reasons of bcl-2 overexpression, but not associated with the accumulation of bcl-2 expressed level during cell transformation.     

Characteristics of chromosome of teratocarcinoma and its influence elements

AIM:To investigate the characteristics of chromosome of teratocarcinoma and its influence elements.METHODS:We used the methods of G-banded karyotype analysis,DNA basic sequence analysis and Western blot and the others,and studied the chromosome karyotype and the status of p53 gene of teratocarcinoma PA-1 cell line which had been cultured for 407-445 passages for 20 years. RESULTS: More than 80% PA-1 cells still maintained the near-diploid karyotype,after passage 30 the cells appeared with M1 and M2 chromosomal markers because of a balanced translocation between chromosomes 15 and 20. DNA directional sequence analysis of RP-PCR products revealed that there were both wild and mutated band (p53 codon 239 mutation), Western blot did not detect mutational p53 gene protein,while p21 protein expression lower than that in normal human fibroblasts. CONCLUSION:Missense mutation of one of the p53 allele gene of PA-1 cells in human teratocarcinoma was detected after cultured for more than twenty years, which was not sufficient to induce the instability of the chromosome of cell line.     

Biological characteristics of primarily cultured microvascular endothelial cells in mouse myocardium

AIM：To investigate an effective and stable method to isolate myocardial microvascular endothelial cells (MMVECs) from the mouse heart and to observe their biological characteristics.METHODS：The cells from the mouse heart were isolated by collagenase II digestion followed by different speed adhesion. Then the cells were cultured on the dish coated with polylysine in endothelial cell-specific culture medium. The biological characteristics were observed by trypan blue staining. The cell growth curve and the cell proliferation were also evaluated by MTT assay at different passages. The expression of DiI-ac-LDL, FITC-UEA-1, specific markers of endothelial cells and the expression of CD31, vWF and CD34 were determined by immunofluoresence staining. To evaluate the function of the MMVECs, in vitro tube formation was evaluated under microscope.RESULTS：Two days after the enzyme digestion, the MMVECs formed small and isolated clusters. The MMVECs grew quickly in monolayer with the characteristics of endothelial cell shape at 4~5 d. The cells became confluent and cobblestone-like which were ready for passage at 7~8 d. After passage, the viability of the cells was more than 95%. The first and the third generation of the cells presented an S-shape growth curve. The cell proliferation of the first to the third generation was quick and slowed down after the fifth passage. MMVECs were highly positive for DiI-ac-LDL and FITC-UEA-1 ［(89.2±3.5)%］, indicating the cells were MMVECs. The other relative antigen expression (CD31, vWF and CD34) on the MMVECs was (56.7±3.7)%, (78.5±2.6)% and (67.8±4.2)%, respectively. The MMVECs formed the tubes in vitro after cultured for 6～12 h.CONCLUSION：We can obtain high-purity MMVECs using the combination of collagenase II digestion, the different speed adhesion process and the endothelial cell-specific culture medium for effective and reliable MMVECs isolation and culture.     

Isolation and culture of rat costochondral growth plate chondrocytes and investigation of their biological character

AIM: To establish the methods for rat costochondral growth plate chondrocyte (RGC) separation and culture and investigate their biological features, thereby provide experimental bases for studying the regulation of chondrocyte proliferation and differentiation. METHODS: RGC were obtained by microdissection and digestion, and cultured in monolayer. Morphological changes of the serial passage of RGC and the cell growth kinectics were observed. The cellular GAG and collagen type Ⅱ expression were detected by histochemistry and ICC. RESULTS: There were more than 98% viable cells in the obtained RGC. The morphology of primary cultured RGC was round or polygon. In this experiment, the sixth passage RGC was still maintained and showed polygonal morphology. The index of duplicatings/day increased in the preceding fourth passage RGC and decreased afterwards. There were more than 95% cells expressed collagen type Ⅱ and alcine blue stained positively in the primary RGC, as the passage number increased, the ratio of collagen type Ⅱ expression and alcine blue positive stained RGC dropped abruptly. CONCLUSION: The separation and culture methods adopted in this study can obtain high pure and viable RGC. The preceding three passage RGC maintains their in vivo phenotype, they are idle experimental materials for studying the regulation of proliferation and differentiation of chondrocyte and tissue engineering.     

Effects of cotransplantation of rat bone marrow mesenchymal ste m cells and bone marrow on hematopoiesis

AIM:To investigate effects of rBMMSC on he matopoiesis and immune reconstitution after allo-hematopoietic stem cells transp lantation (HSCT).METHODS:Allogeneic BMT model from Fischer344 rats (RT-1Al) to W istar rats (RT-1Au) was established.The effects of MSCs on hematopoietic recons titution and immune reconstitution were studied by observing the survival rate,peripheral blood counts,thymus counts,spleen counts,bone marrow counts and im mune function analysis at 30 days after transplantation.RESULTS:1.Cotransplantation of MSCs and bone marrow (BM) was d emonstrated to improve hematopoietic reconstitution.Lymphocyte and platelet cou nts in peripheral blood in cotransplantation groups were higher than those in co ntrol groups.More bone marrow neucleated cells were also observed in cotranspla ntation groups.2.Cotransplantation of MSCs and BM improved immune reconstituti on.First,overall thymic cellularity and spleen cellularity significantly incre ased in cotransplantation groups at day 30.Secondly,cotransplantation improved immune functional recovery.Non-specific lymphocytes proliferation reaction ind uced by ConA and LPS increased in cotransplantation group,and so did for alloge neic mixed-lymphocyte reaction.CONCLUSION:Hematopoietic reconstitution and immune reconstituti on were significantly enhanced by MSCs cotransplanted with BM.     

Effect of continuous subculturing of hUC-MSCs on mRNA expression of NLR family

AIM：To investigate the influence of continuous subculturing of human umbilical cord mesenchymal stem cells (hUC-MSCs) on the mRNA expression of all 23 family members of NOD-like receptors (NLRs), and to search for the way of improving the subculture quality of hUC-MSCs and increasing the quantity and safety in the experimental and clinical application. METHODS：Neonatal umbilical cord was collected to isolate and purify the hUC-MSCs with the collagenase II digestion and adherence screening methods. These cells were continuously subcultured. The hUC-MSCs at passage 3 and passage 28 were identified by flow cytometry and induced differentiation. The mRNA expression of NLRs in the passage 3 and passage 28 hUC-MSCs was detected by RT-qPCR. RESULTS：The cell phenotypes of both passage 3 and passage 28 hUC-MSCs were CD29+/CD44+/CD105+/ CD31-/CD34-/CD40-/CD45-/CD106-/HLA-DR-, and both of the cells were induced into osteoblasts and adipocytes, which were conformed to the criteria of International Society for Cellular Therapy to define MSCs. All the NLR family members were expressed in passage 3 hUC-MSCs. NOD1, NLRC4, NLRC5, NLRP1, NLRP3, NLRP10, NAIP, NLRX1 and APAF1 at mRNA levels were highly expressed, and the rest were lowly expressed. When hUC-MSCs were subcultured to passage 28, NLRP10 mRNA was increased, NLRC5 mRNA and NLRX1 mRNA were hardly changed, and all of the rest members were decreased. The difference of NLRP1 mRNA expression between passage 3 and passage 28 hUC-MSCs was observed with statistical significance (P<0.05). CONCLUSION：The effects of subculturing on the expression of NLR family in hUC-MSCs are pleiotropic. It requires further investigation to confirm whether these effects are related to the proliferation, differentiation and immunomodulation of MSCs.     

Establishment of patient-derived esophageal squamous-cell carcinoma xenograft in mice and characteristics of signaling pathways related to proliferation in SCID mice

AIM: To establish and characterize the patient-derived esophageal squamous-cell carcinoma xenograft (PDECX) in mice. METHODS: The samples of human esophageal cancer were grafted into severe combined immunodeficient (SCID) mice. The xenografts were transferred to SCID mice when the first passage of xenografts grew up. The growth of tumors in the first, second and third passages was observed. HE staining was performed. The expression of CK5/6, p63 and p40 in the patient samples, and the first and third passages of the xenografts were detected by immunohistochemical analysis. The expression of mTOR, p-mTOR, p70S6K, p-p70S6K, Akt1, p-Akt (Ser473), Erk1/2 and p-Erk1/2 were determined by Western blot.RESULTS: The PDECX was successfully established. The positive expression of CK5/6, p63 and p40 in the xenografts was consistent with that in the patients' samples. The levels of phosphorylated and total proteins of proliferation-related signaling pathways were different in the xenografts from different patients.CONCLUSION: The PDECX model adequately reflects the tumal heterogeneity that is observed in the patients.     

Rat mesenchymal stem cells differentiate into neuron-like cells with adrenaline hormones

AIM: To investigate the differentiation from rat mesenchymal stem cells (rMSC) into neuron-like cells. METHODS:rMSC were separated from femur marrow and expanded in L-DMEM culture medium supplemented with 10% FSC. rMSC were induced to differentiate into neurons with L-DMEM/adrenaline,L-DMEM/noradrenaline and L-DMEM/isoprenaline, respectively. Meanwhile, rMSC were cultured in L-DMEM in control group. Nestin, neuron-specific enclose (NSE), glial fibrillary acidic protein (GFAP) were detected by immunocytochemistry. RESULTS: rMSC were expanded as undifferentiated cells in culture from 5 to 22 passages, indicating their differentiated capacity. Simple method induced rMSC to exhibit a neuronal phenotype, expressing positive NSE,nestin, and GFAP, at 5 hours in all group. The undifferentiating cells (control group 53.1%±4.3%), and differentiating cells (treated group: adrenaline 74.7%±2.6%; noradrenaline 75.9%±2.4%; isoprenaline 72.1%±4.4%), expressed characteristics of various neuronal cells, from 5 hours to 6 days. There were neuron-like cells in rMSC cultured in L-DMEM/10%FBS from 7 to 13 passage(66.5%±6.4%). CONCLUSION: It suggests that rat neural stem cells (rNSC) exist in bone marrow, rMSC can be differentiated into various neural cells with adrenaline hormones in vitro.     

Rapid proliferation of bone marrow mesenchymal stem cells enhanced by xanthosine

AIM: To explore the effect of xanthosine (Xs) on proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs). METHODS: Xs was directly added to the culture system and the effects of Xs on proliferation and differentiation of BMSCs were observed. RESULTS: In the presence of Xs, the growth rate of the 10th passage cells was almost similar to that of the 4th passage cells and no downtrend was observed. However, in control group (without Xs), the growth rate of the 10th passage cells was obviously declined. The BMSCs promoted by Xs still kept up a vigorous capability of differentiation into hepatocytes. CONCLUSION: As an inhibitor of asymmetric cell kinetics, Xs can promote the conversion of BMSCs from asymmetric cell kinetics to symmetric cell kinetics, and keeps synchronously the ability of differentiation from BMSCs into hepatocytes as well. It is helpful for enhancing the proliferation efficiency of BMSCs in vitro, and will have extensive application of clinical practice.     

Effects of bradykinin on proliferation of porcine pulmonary artery smooth muscle cells induced by TGF-β1

AIM: To investigate the effects of bradykinin (BK) on the proliferation of pulmonary artery smooth muscle cells (PASMCs) induced by transforming growth factor beta 1 (TGF-β1) and its possible mechanisms. METHODS: Primary porcine PASMCs were isolated, cultured and identified, and the cells at passages 2~6 were used in this study. The viability of PASMCs was determined by Cell Counting Kit-8 assay. The protein expression of phosphatidylinositol 3-kinase (PI3K), phosphorylated Akt (p-Akt) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) was detected by Western blotting. RESULTS: TGF-β1 promoted the proliferation of PASMCs in a dose-dependent manner (P<005). BK significantly inhibited the proliferation of PASMCs induced by TGF-β1 (P<005), and attenuated the elevated expression of PI3K, p-Akt and p-ERK1/2 proteins (P<005). HOE-140, a BK type 2 receptor (B2R) inhibitor, reversed the effects of BK (P<005). CONCLUSION: BK inhibits TGF-β1-induced proliferation of PASMCs, which may be associated with inactivation of PI3K/Akt and ERK1/2 signaling pathways.     

Ectopic hTERT gene expression in human bone marrow mesenchymal stem cells

AIM: To investigate the effect of transfection of hTERT gene into human mesenchymal stem cells (MSCs) on their telomerase activity and life-span.METHODS: Human MSCs were transfected with a pEGFP-hTERT plasmid by liposome-mediated transfection. Then the hTERT mRNA expression in MSCs was detected by RT-PCR. The activity of telomerase in transfected MSCs was detected by PCR and ELISA. The telomerase-positive MSCs was cultured in vitro and induced into neuron-like cells with EGF and bFGF. Neuron-specific markers (NF-M, MAP₂) were detected by RT-PCR.RESULTS: hTERT fragment was identified in the hTERT-transfeced cells but not in the untransfected human bone marrow MSCs. The untransfected human MSCs remained telomerase-negative but the hTERT-transfected cells showed robust telomerase activity. The telomerase-negative MSCs entered a nondividing state and senesced after about 20 to 25 passages. In test group, however, telomerase-positive MSCs to date had undergone 35 passages. RT-PCR analysis showed that telomerase-positive MSCs expressed neuron-specific markers, such as NF-M or MAP₂ after induced with EGF and bFGF in vitro. CONCLUSION: Ectopic expression of the hTERT gene in human MSCs reconstitutes telomerase activity. The transfection of hTERT gene into human MSCs extends their replicative life span and maintains their multipotent differentiation capacity.     

Study on the culture of rat myocardium microvascular endothelial cells and microarray analysis

AIM: To study the cytological characteristics of rat myocardium microvascular endothelial cells (RMMVEC) by microarray. METHODS: The RMMVEC were cultured by the method of planting myocardium tissue. The morphology of RMMVEC was studied by light and electronic microscopy. Its molecular markers were observed by immunocytochemistry. Cell proliferation kinetic was analyzed by counting the number of cells. The gene expression of the RMMVEC was studied by endothelial cell biology gene microarray and compared the change of gene expression among the cultured cells of primary, 2nd and 5th passage. RESULTS: The RMMVEC showed morphological characteristics of microvascular endothelial cells (MVEC): growing in a cobblestone pattern, forming tube-like structure or capillary network and having microvilli on cell surface. At the same time, the RMMVEC showed positive staining for vWF, CD34, CD31, CD105 and Tie-2. Gene microarray analysis indicated expression of VEGFR, ICAM-1, VCAM-1, angiopoietin1, PECAM1 (CD31) and other genes closely related to microvascular endothelial functions at relatively high level. But in cultured cells of 5th passage the characteristic gene expression of microvascular endothelial cells disappeared. CONCLUSION: The RMMVEC cultured by this method possess typical characteristics of MVEC. The cytological characteristics are steady in the cultured cells of primary and 2nd passage. It can be utilized to study the mechanisms of some cardiovascular diseases.     

Purification and adipogenic differentiation of human yolk sac mesenchymal stem cells

AIM:To purify human yolk sac mesenchymal stem cells (hYS-MSC) and investigate its adipogenic differentiation potential. METHODS:hYS-MSC were separated from yolk sac and purified via passaged culture. Flow cytometric analysis was used to identify the phenotype of hYS-MSC and the alkaline phosphatase(AKP) expression of hYS-MSC was also tested. Adipogenic differentiation of hYS-MSCs was induced by 10 mg/L insulin, 10^-5mol/L indomethacin and 10^-6mol/L dexamethasone. Oil Red O was used for fat staining. RESULTS:hYS-MSCs were purified at passages 2 or 3. Flow cytometric analysis showed the phenotype of purified YS-MSCs was uniformly positive for CD29, CD44, CD105, and CD166, and negative for reactivity to antigens CD34, CD45, or CD86. hYS-MSCs were weakly but clearly positive in AKP. Adipogenic differentiation of YS-MSCs was induced by 10 mg/L insulin, 10^-5mol/L indomethacin and 10^-6mol/L dexamethasone. Accumulation of lipid-rich vacuoles positive in oil red O staining within the cells were appeared and nuclears were pushed to one side of the cells during the period of induction. CONCLUSION:The phenotype of hYS-MSC is coincident with adult human mesenchymal stem cells. hYS-MSC can be induced to differentiate into adipocytes in vitro.