The Mechanism, Regulatory Effect of Wnt5α/Ca2+ Signaling Pathway on hMSCs Osteogenic Differentiation Under Infection Conditions

2019 ◽  
Vol 9 (12) ◽  
pp. 1706-1711
Author(s):  
Yilizati Yilihamu ◽  
Liu Yansong ◽  
Xu Mingliang ◽  
Cao Tianyong ◽  
Shi Rongjian
Keyword(s):  
Osteogenic Differentiation ◽  
Signaling Pathway ◽  
Calcium Concentration ◽  
Wnt Signaling Pathway ◽  
Quantitative Detection ◽  
Control Group ◽  
Specific Gene ◽  
Beta Catenin ◽  
Alizarin Red

Background: The paper aimed to reveal the role and possible regulatory mechanisms of Wnt5 gene and its non-canonical Beta-catenin signaling pathway in osteogenic differentiation and bone repairment of hMSCs under infection environment. Material and Methods : CCK-8 method was employed to detect the effect of SPA on the proliferation of different hMSCs cells. The histological staining of alizarin red was used to observe the differentiation of cells into osteoblasts. The semi-quantitative detection of osteoblast alkaline phosphatase (ALP) was performed. Fluo-2 assay was employed to measure the intracellular calcium concentration of different hMSCs before and after osteogenic differentiation stimulated by SPA. RT-PCR was applied to detect the specific gene fluorescence of osteoblasts differentiation. Western Blotting was employed to determine the differentiation of hMSCs into osteoblasts. Results: Different hMSCs were able to proliferate in vitro under the stimulation of 100 ng/mL SPA. CCK-8 kit was used to detect lentivirus-infected hMSCs and non-transduced hMSCs in vitro. The results showed that all the cells gradually proliferate with time and an increasing rate. The hMSCs treated with different Wnt5 genes were stained with alizarin red after osteogenic differentiation, and the activities of AMSCs ALP increased. The intracellular free calcium concentration increased with the induction time and peaked in the Wnt5 overexpression group. PCR results showed that ALP, Runx-2, Collagen I, OC, Osterix and BSP genes were all increased in contrast with the non-induction Control group. Conclusion: Under the inflammatory condition of SPA virulence factors, non-canonical Wnt signaling pathway is associated with the classical Wnt/ β-catenin signaling pathway in the process of osteogenic differentiation of hMSCs in vitro, which has a certain synergistic effect.

scholarly journals Knockdown of POSTN Inhibits Osteogenic Differentiation of Mesenchymal Stem Cells From Patients With Steroid-Induced Osteonecrosis

2020 ◽  
Vol 8 ◽  
Author(s):  
Lizhi Han ◽  
Song Gong ◽  
Ruoyu Wang ◽  
Shaokai Liu ◽  
Bo Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Bone Formation ◽  
Osteogenic Differentiation ◽  
Control Group ◽  
Nodule Formation ◽  
Matricellular Protein ◽  
Specific Gene ◽  
Endogenous Expression

Steroid-induced osteonecrosis of femoral head (SONFH) is a common and serious complication caused by long-term and/or excessive use of glucocorticoids (GCs). The decreased activity and abnormal differentiation of bone marrow mesenchymal stem cells (BMSCs) are considered to be one of the major reasons for the onset and progression of this disease. Periostin (POSTN) is a matricellular protein which plays an important role in regulating osteoblast function and bone formation. Sclerostin (SOST) is a secreted antagonist of Wnt signaling that is mainly expressed in osteocytes to inhibit bone formation. However, the exact role of POSTN and SOST in SONFH has not been reported yet. Therefore, we detected the differential expression of POSTN and SOST in BMSCs of SONFH Group patients, and Control Group was patients with traumatic ONFH (TONFH) and developmental dysplasia of the hip (DDH). Furthermore, we used lentiviral transfection to knockdown POSTN expression in BMSCs of patients with SONFH to study the effect of POSTN knockdown on the SOST expression and osteogenic differentiation of BMSCs. The results indicated that the endogenous expression of POSTN and SOST in BMSCs of SONFH Group was upregulated, compared with Control Group. POSTN was upregulated gradually while SOST was downregulated gradually at days 0, 3, and 7 of osteogenic differentiation of BMSCs in Control Group. Contrarily, POSTN was gradually downregulated while SOST was gradually upregulated during osteogenic differentiation of BMSCs in SONFH Group. This could be due to increased expression of SOST in BMSCs, which was caused by excessive GCs. In turn, the increased expression of POSTN in BMSCs may play a role in antagonizing the continuous rising of SOST during the osteogenic differentiation of BMSCs in patients with SONFH. POSTN knockdown significantly attenuated osteo-specific gene expression, alkaline phosphatase activity, and calcium nodule formation in vitro; thus inhibiting the osteogenic differentiation of BMSCs in patients with SONFH. Besides, POSTN knockdown upregulated SOST expression, increased GSK-3β activity, and downregulated β-catenin. These findings suggest that POSTN have an essential role in regulating the expression of SOST and osteogenic differentiation of BMSCs in patients with SONFH, and POSTN knockdown suppresses osteogenic differentiation by upregulating SOST and partially inactivating Wnt/β-catenin signaling pathway. Therefore, targeting POSTN and SOST may serve as a promising therapeutic target for the prevention and treatment of SONFH.

Downregulation of SATB1 Increase the Invasiveness of Jurkat Cell Via Activation of the WNT/Beta-Catenin Signaling Pathway in Vitro

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4825-4825
Author(s):  
Xiaodan Luo ◽  
Lihua Xu ◽  
Dan Liu ◽  
Yaya Wang ◽  
Xiaohong Wu ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Peripheral Blood ◽  
Jurkat Cell ◽  
Cellular Proliferation ◽  
Conflicts Of Interest ◽  
Expression Patterns ◽  
Control Group ◽  
Beta Catenin ◽  
Normal Controls

Abstract Backgroud: Special AT-rich sequence-binding protein-1 (SATB1) is critical for genome organizer that reprograms chromatin organization and transcription profiles, and associated with tumor growth and metastasis in several cancer types. Many studies suggest that SATB1 overexpression is an indicator of poor prognosis in various cancers, such as breast cancer, malignant cutaneous melanoma, liver cancer, etc. However, their expression patterns and function values for adult T-cell leukemia (ATL) are still largely unknown. Objective: The aim of this study is to examine the levels of SATB1 in ATL and to explore its function and mechanisms in ATL. METHODS: 20 ATL peripheral blood samples and 20 normal controls were collected. Expressions of SATB1 in both groups were evaluated by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Cellular proliferation and invasion of SATB1-knockdown Jurkat cells and cells in control group were evaluated by manually count and transwell matrigel invasion assay, respectively. RESULTS: SATB1 expressions were decreased in ATL peripheral blood mononuclear cells (p<0.001) compared with normal controls. Knockdown of SATB1 gene might increase Jurkat cell invasiveness through the activation of Wnt/β-Catenin signaling pathway. CONCLUSIONS: SATB1 expression is down-regulated in ATL and decreased expression of SATB1 increase the invasiveness of Jurkat cell through the activation of Wnt/β-Catenin signaling pathway in vitro. Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (81200399) and Key Clinical Disciplines of Guangdong Province (20111219) Disclosures No relevant conflicts of interest to declare.

scholarly journals Icariin Protects against Glucocorticoid-Induced Osteonecrosis of the Femoral Head in Rats

2018 ◽  
Vol 47 (2) ◽  
pp. 694-706 ◽  
Author(s):  
Zengfa Huang ◽  
Cheng Cheng ◽  
Beibei Cao ◽  
Jing Wang ◽  
Hui Wei ◽  
...  
Keyword(s):  
Femoral Head ◽  
Osteogenic Differentiation ◽  
Bone Repair ◽  
Adipogenic Differentiation ◽  
Ct Scanning ◽  
Control Group ◽  
Micro Ct ◽  
Alizarin Red ◽  
Immunohistochemical Analyses

Background/Aims: Glucocorticoid (GC)-related osteonecrosis of the femoral head (ONFH) is a common complication following administration of steroids to treat many diseases. Our previous study demonstrated that icariin (ICA) might have a beneficial effect on the bone marrow mesenchymal stem cells (BMSCs) of patients with steroid-associated osteonecrosis. In this study, we investigated the underlying mechanisms of ICA associated with the potential enhancement of osteogenesis and anti-adipogenesis in GC-related ONFH. Methods: In vitro cell proliferation was evaluated by CCK-8 assay. Alizarin red S and alkaline phosphatase (ALP) activity were used to measure osteogenic differentiation, while adipogenic differentiation was revealed by oil red O staining and TG content assay. The expression level of osteogenesis-associated genes and PPARγ was evaluated by RT-qPCR, western blotting and immunofluorescence. A total of 30 female SD rats were randomly separated into three groups: a control group, a methylprednisolone (MPS) group and a MPS + ICA group. Serum ALP and TG (triglyceride), micro-CT scanning, histological and immunohistochemical analyses were performed in the animal model. Results: In the in vitro study, ICA promoted proliferation, improved osteogenic differentiation and suppressed adipogenic differentiation of BMSCs treated with MPS. The group treated with MPS and 10-6 M ICA expressed higher levels of Runx2, ALP, bone morphogenetic protein (BMP) 2, and OC and lower expression of PPARγ than the MPS group. In the in vivo study, ICA prevented bone loss in a rat model of GC-related ONFH as shown by micro-CT scanning, histological and immunohistochemical analyses. Conclusions: ICA is an effective compound for promoting bone repair and preventing or delaying the progression of GC-associated ONFH in rats. This effect can be explained by its ability to improve the balance between adipogenesis and osteogenesis, indicating that ICA is an effective candidate for management of GC-associated ONFH.

scholarly journals Effects of Concentrated Growth Factor on the Proliferation, Migration, and Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells: An in vitro study

2020 ◽  
Author(s):  
Xiang Yu ◽  
Hui Ren ◽  
Qi Shang ◽  
Gengyang Shen ◽  
Kai Tang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Osteogenic Differentiation ◽  
Control Group ◽  
Elisa Assay ◽  
Alizarin Red ◽  
Expression Levels ◽  
Marrow Mesenchymal Stem Cells ◽  
Mrna Expression Levels

Abstract Background Concentrated growth factor (CGF) has been reported to be effective in bone formation or soft/hard tissue healing in recent years. Despite a few studies regarding the effects of CGF on the proliferation, migration, and osteogenic differentiation of BMSCs, their underlying mechanisms are not fully understood. The purpose of this study is to investigate the effects and possible mechanisms of CGF on the proliferation, migration, and osteogenic differentiation of rat-derived bone marrow mesenchymal stem cells (BMSCs) in vitro. Methods CGF was extracted from the Sprague Dawley (SD) rats by venipuncture of the abdominal aortic vein, and scanning electron microscopy (SEM) was used for the structural characterization. The release of bone morphogenetic protein 2 (BMP-2) from CGF was measured over the periods of 1 ~ 14 days, using the enzyme-linked immunosorbent (Elisa) assay. Cell Counting Kit-8 (CCK-8) assay was used to measure cell proliferation. Migration capacity was analyzed using the transwell assay. The osteogenic differentiation and mineralization ability were determined by Alkaline phosphatase activity (ALP) staining and Alizarin Red staining respectively. Quantitative real-time PCR (RT-qPCR), was used to evaluate the mRNA expression levels of Runx2, Ocn, Smad1, and Smad5 after culture for 14 days. Further, the protein expression of BMP-2, phosphorylated-Smad1/5 (p-Smad1/5), and Smad1/5/8 was determined by Western blot after a 14-day cell culture. Results The SEM analysis showed a porous and dense three-dimensional fibrin network in CGF. The Elisa assay showed that BMP-2 was released from CGF extract for more than 14d, and it reached a peak at the time point of 5d. The cell densities of the CGF group at the different concentrations (5%, 10%, and 20%) were significantly higher than that of the control group at the periods of day 1 to day 5 (p < 0.05). Moreover, the number of migratory cells of the CGF group was greater than that of the control group at 24 h. ALP activity analysis and Alizarin Red staining results demonstrated that CGF may successfully induce osteogenic differentiation of BMSCs. Moreover, the RT-qPCR results showed that CGF extracts dramatically enhanced the mRNA expression levels of Runx2, Ocn, Smad1, and Smad5 in BMSCs at days 14 (p < 0.05). Furthermore, Western blot results showed that CGF extracts markedly up-regulated the protein expression levels of BMP-2, p-Smad1/5, and Smad1/5/8. Conclusions CGF can promote the proliferation, migration, and promote the osteogenic differentiation potential of BMSCs in vitro. The BMP-2/Smad signaling pathway was involved in the osteogenic differentiation and mineralization of BMSCs induced by CGF. Therefore, CGF has good application potential in tissue engineering for bone regeneration and repair.

scholarly journals Role of Fzd6 in Regulating the Osteogenic Differentiation of Adipose-derived Stem Cells in Osteoporosis

2021 ◽  
Author(s):  
Tianli Wu ◽  
Zhihao Yao ◽  
Gang Tao ◽  
Fangzhi Lou ◽  
Hui Tang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Wnt Signaling Pathway ◽  
Osteogenic Potential ◽  
Adipose Derived Stem Cells ◽  
Alizarin Red ◽  
Differentiation Potential

Abstract Objective: Although it has been demonstrated that adipose-derived stem cells (ASCs) from osteoporosis mice (OP-ASCs) exhibit impaired osteogenic differentiation potential, the molecular mechanism has not yet been elucidated. We found that Fzd6 was decreased in OP-ASCs compared with ASCs. This study investigates the effects and underlying mechanisms of Fzd6 in the osteogenic potential of OP-ASCs. Methods: Fzd6 expression in ASCs and OP-ASCs was measured by PCR gene chip. Fzd6 overexpression and silencing lentiviruses were used to evaluate the role of Fzd6 in the osteogenic differentiation of OP-ASCs. Real-time PCR (qPCR) and western blotting (WB) was performed to detect the expression of Fzd6 and bone-related molecules, including runt-related transcription factor 2 (Runx2) and osteopontin (Opn). Alizarin red staining and Alkaline phosphatase (ALP) staining was performed following osteogenic induction. Microscopic CT (Micro-CT), hematoxylin and eosin staining (H&E) staining, and Masson staining were used to assess the role of Fzd6 in osteogenic differentiation of osteoporosis (OP) mice in vivo.Results: Expression of Fzd6 was decreased significantly in OP-ASCs. Fzd6 silencing down-regulated the osteogenic ability of OP-ASCs in vitro. Overexpression of Fzd6 rescued the impaired osteogenic capacity in OP-ASCs in vitro. We obtained similar results in vivo.Conclusions: Fzd6 plays an important role in regulating the osteogenic ability of OP-ASCs both in vivo and in vitro. Overexpression of Fzd6 associated with the Wnt signaling pathway promotes the osteogenic ability of OP-ASCs, which provides new insights for the prevention and treatment of OP.

scholarly journals Baicalein Accelerates Tendon-Bone Healing via Activation of Wnt/β-Catenin Signaling Pathway in Rats

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Xinggui Tian ◽  
Huaji Jiang ◽  
Yuhui Chen ◽  
Xiang Ao ◽  
Chuan Chen ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Signaling Pathway ◽  
Bone Healing ◽  
Tendon Graft ◽  
Bone Tunnel ◽  
Control Group ◽  
Differentiation Markers ◽  
Related Transcription Factor

Background. Tendon-bone healing is a reconstructive procedure which requires a tendon graft healing to a bone tunnel or to the surface of bone after the junction injury between tendon, ligament, and bone. The surgical reattachment of tendon to bone often fails due to regeneration failure of the specialized tendon-bone junction. Materials and Methods. An extra-articular tendon-bone healing rat model was established to discuss the effect of the baicalein 10 mg/(kg·d) in accelerating tendon-bone healing progress. Also, tendon-derived stem cells (TDSCs) were treated with various concentrations of baicalein or dickkopf-1 (DKK-1) to stimulate differentiation for 14 days. Results. In vivo, tendon-bone healing strength of experiment group was obviously stronger than the control group in 3 weeks as well as in 6 weeks. And there were more mature fibroblasts, more Sharpey fibers, and larger new bone formation area treated intragastrically with baicalein compared with rats that were treated with vehicle for 3 weeks and 6 weeks. In vitro, after induction for 14 days, the expressions of osteoblast differentiation markers, that is, alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osteocalcin (OCN), osterix (OSX), and collagen I, were upregulated and Wnt/β-catenin signaling pathway was enhanced in TDSCs. The effect of DKK-1 significantly reduced the effect of baicalein on the osteogenic differentiation. Conclusion. These data suggest that baicalein may stimulate TDSCs osteogenic differentiation via activation of Wnt/β-catenin signaling pathway to accelerate tendon-bone healing.

186 The effects of different concentrations of MgSO4 in osteogenic differentiation

2019 ◽  
Vol 31 (1) ◽  
pp. 217
Author(s):  
L. R. Padoveze ◽  
M. Rubessa ◽  
C. E. Ambrosio ◽  
M. B. Wheeler
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
In Vitro Study ◽  
Control Group ◽  
Nodule Formation ◽  
Osteogenic Medium ◽  
Alizarin Red ◽  
Physiological Processes

Tissue engineering offers a viable alternative to bone grafts in repairing large bone defects. Magnesium-based materials are biocompatible in vivo, and it is possible to determine the degradation period according to the necessities (Farraro et al. 2014 J. Biomech. 47, 1979-1986). Magnesium (Mg) is part of many physiological processes, and it promotes the osteogenesis of mesenchymal stem cells (Díaz-Tocados et al. 2017 Sci. Rep. 7, 7839.). Moreover, Mg up-regulates important genes associated with the osteogenic differentiation (Yoshizawa et al. 2014 Acta Biomater. 10, 2834-2842). The aim of this study was to evaluate the effect of different Mg concentrations in the osteogenic medium on the number of nodules of bone. Swine adipose stem cells (ASC) were previously isolated as described (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20-33). In this in vitro study, ASC were cultured during 4 weeks in osteogenic medium with addition of 0.1, 0.2, 1, 2, 10, or 20mM MgSO4. The medium was changed twice a week. Alizarin Red and Von Kossa staining were performed to evaluate the formation of nodules by mineralization of extracellular matrix (ECM), evidenced by dark red nodules and calcium deposit. The experiment was replicated 3 times in triplicate. Data were analysed using the generalized linear model (GLM) procedure, and Bonferroni’s post hoc test was used to perform statistical multiple comparison (SPSS Inc./IBM Corp., Chicago, IL, USA). The results showed enhanced nodule formation with 2mM Mg in the osteogenic medium (35.6v. 15.3, respectively for 2mM and Control). This result confirms the ability of magnesium to act in bone formation. There was no statistical difference among the different groups when we evaluated the Von Kossa staining results, indicating that the quality of the new formations was comparable to that of the control group even in an elevated nodule formation. In conclusion, a higher concentration of magnesium can improve nodule formation into osteogenic differentiation in vitro; the 2mM concentration showed the best nodule formation compared with the other groups. These results showed the value of magnesium in bone physiology.

Synthesis and characterization of melatonin-loaded chitosan microparticles promote differentiation and mineralization in pre-osteoblastic cells

2020 ◽  
Author(s):  
Ren-Yeong Huang ◽  
Po-Yan Hsiao ◽  
Lian-Ping Mau ◽  
Yi-Wen Cathy Tsai ◽  
David L. Cochran ◽  
...  
Keyword(s):  
Sustained Release ◽  
Osteogenic Differentiation ◽  
Control Group ◽  
Alizarin Red ◽  
Positive Effects ◽  
Related Transcription Factor ◽  
Release Assay ◽  
Investigation Period ◽  
Novel Scaffold

In terms of a novel scaffold with well osteoinductive and osteoconductive capacity, melatonin (Mel) possesses positive effects on chemical linkage in scaffold structures, which may allow osteogenic differentiation.  The aim of this study is to fabricate Mel-loaded chitosan (CS) microparticles (MPs) as a novel bone substitute through generating a Mel sustained release system from Mel-loaded CS MPs, and evaluating its effect on the osteogenic capacity of MC3T3-E1 in vitro.  The physical-chemical characteristics of the prepared CS MPs were examined by both Fourier transform infrared spectroscopy and scanning electron microscopy.  The released profile and kinetics of Mel from MPs were quantified, and the bioactivity of the released Mel on pre-osteoblasts MC3T3-E1 cells was characterized in vitro.  In vitro drug release assay has shown high encapsulation efficiency and sustained release of Mel over the investigation period.  In an osteogenesis assay, Mel-loaded CS MPs have significantly enhanced Alkaline phosphatase (Alp) mRNA expression and ALP activity compared to the control group.  Meanwhile, the osteoblast specific differentiation genes, including runt related transcription factor 2 (Runx2), bone morphogentic protein-2 (Bmp2), and collagen I (Col I) and osteocalcin (Ocn) were also significantly up-regulated.  Furthermore, quantificational alizarin red-based assay demonstrated that Mel-loaded CS MPs notably enhanced the calcium deposit of MC3T3-E1 compared to the controls.  In essence, Mel-loaded CS MPs can control the release of Mel for a period of time to accelerate osteogenic differentiation of pre-osteoblast cells in vitro.

scholarly journals MicroRNA-1297 suppressed the Akt/GSK3β signaling pathway and stimulated neural apoptosis in an in vivo sevoflurane exposure model

2021 ◽  
Vol 49 (4) ◽  
pp. 030006052098210
Author(s):  
Quan Wang ◽  
Jingcong Luo ◽  
Ruiqiang Sun ◽  
Jia Liu
Keyword(s):  
Protein Expression ◽  
Signaling Pathway ◽  
Neuronal Apoptosis ◽  
In Vitro Model ◽  
Nervous System Development ◽  
Exposure Model ◽  
Control Group ◽  
Pten Protein ◽  
Vitro Model

Objective Common inhalation anesthetics used for clinical anesthesia (such as sevoflurane) may induce nerve cell apoptosis during central nervous system development. Furthermore, anesthetics can produce cognitive impairments, such as learning and memory impairments, that continue into adulthood. However, the precise mechanism remains largely undefined. We aimed to determine the function of microRNA-1297 (miR-1297) in sevoflurane-induced neurotoxicity. Methods Reverse transcription-polymerase chain reaction assays were used to analyze miR-1297 expression in sevoflurane-exposed mice. MTT and lactate dehydrogenase (LDH) assays were used to measure cell growth, and neuronal apoptosis was analyzed using flow cytometry. Western blot analyses were used to measure PTEN, PI3K, Akt, and GSK3β protein expression. Results In sevoflurane-exposed mice, miR-1297 expression was up-regulated compared with the control group. MiR-1297 up-regulation led to neuronal apoptosis, inhibition of cell proliferation, and increased LDH activity in the in vitro model of sevoflurane exposure. MiR-1297 up-regulation also suppressed the Akt/GSK3β signaling pathway and induced PTEN protein expression in the in vitro model. PTEN inhibition (VO-Ohpic trihydrate) reduced PTEN protein expression and decreased the effects of miR-1297 down-regulation on neuronal apoptosis in the in vitro model. Conclusion Collectively, the results indicated that miR-1297 stimulates sevoflurane-induced neurotoxicity via the Akt/GSK3β signaling pathway by regulating PTEN expression.

scholarly journals Establishing a deeper understanding of the osteogenic differentiation of monolayer cultured human pluripotent stem cells using novel and detailed analyses

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ping Zhou ◽  
Jia-Min Shi ◽  
Jing-E Song ◽  
Yu Han ◽  
Hong-Jiao Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Telomerase Activity ◽  
Human Pluripotent Stem Cells ◽  
Cell Counting ◽  
Cell Type ◽  
Alizarin Red

Abstract Background Derivation of osteoblast-like cells from human pluripotent stem cells (hPSCs) is a popular topic in bone tissue engineering. Although many improvements have been achieved, the low induction efficiency because of spontaneous differentiation hampers their applications. To solve this problem, a detailed understanding of the osteogenic differentiation process of hPSCs is urgently needed. Methods Monolayer cultured human embryonic stem cells and human-induced pluripotent stem cells were differentiated in commonly applied serum-containing osteogenic medium for 35 days. In addition to traditional assays such as cell viability detection, reverse transcription-polymerase chain reaction, immunofluorescence, and alizarin red staining, we also applied studies of cell counting, cell telomerase activity, and flow cytometry as essential indicators to analyse the cell type changes in each week. Results The population of differentiated cells was quite heterogeneous throughout the 35 days of induction. Then, cell telomerase activity and cell cycle analyses have value in evaluating the cell type and tumourigenicity of the obtained cells. Finally, a dynamic map was made to integrate the analysis of these results during osteogenic differentiation of hPSCs, and the cell types at defined stages were concluded. Conclusions Our results lay the foundation to improve the in vitro osteogenic differentiation efficiency of hPSCs by supplementing with functional compounds at the desired stage, and then establishing a stepwise induction system in the future.

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