Influence of the addition of nanohydroxyapatite to scaffolds on proliferation and differentiation of human mesenchymal stem cells: a systematic review of in vitro studies

One of the main challenges of tissue engineering in dentistry is to replace bone and dental tissues with strategies or techniques that simulate physiological tissue repair conditions. This systematic review of in vitro studies aimed to evaluate the influence of the addition of nanohydroxyapatite (NHap) to scaffolds on cell proliferation and osteogenic and odontogenic differentiation of human mesenchymal stem cells. In vitro studies on human stem cells that proliferated and differentiated into odontogenic and osteogenic cells in scaffolds containing NHap were included in this study. Searches in PubMed/MEDLINE, Scopus, Web of Science, OpenGrey, ProQuest, and Cochrane Library electronic databases were performed. The total of 333 articles was found across all databases. After reading and analyzing titles and abstracts, 8 articles were selected for full reading and extraction of qualitative data. Results showed that despite the large variability in scaffold composition, NHap-containing scaffolds promoted high rates of cell proliferation, increased alkaline phosphatase (ALP) activity during short culture periods, and induced differentiation, as evidenced by the high expression of genes involved in osteogenesis and odontogenesis. However, further studies with greater standardization regarding NHap concentration, type of scaffolds, and evaluation period are needed to observe possible interference of these criteria in the action of NHap on the proliferation and differentiation of human stem cells.

Culture of rat mesenchymal stem cells on PHBV-PCL scaffolds: analysis of conditioned culture medium by FT-Raman spectroscopy / Cultivo de células-tronco mesenquimais de rato sobre arcabouços de PHBV-PCL: análise do meio de cultura condicionado por espectroscopia de FT-Raman

Abstract Mesenchymal stem cells (MSCs) have great potential for application in cell therapy and tissue engineering procedures because of their plasticity and capacity to differentiate into different cell types. Given the widespread use of MSCs, it is necessary to better understand some properties related to osteogenic differentiation, particularly those linked to biomaterials used in tissue engineering. The aim of this study was to develop an analysis method using FT-Raman spectroscopy for the identification and quantification of biochemical components present in conditioned culture media derived from MSCs with or without induction of osteogenic differentiation. All experiments were performed between passages 3 and 5. For this analysis, MSCs were cultured on scaffolds composed of bioresorbable poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL) polymers. MSCs (GIBCO®) were inoculated onto the pure polymers and 75:25 PHBV/PCL blend (dense and porous samples). The plate itself was used as control. The cells were maintained in DMEM (with low glucose) containing GlutaMAX® and 10% FBS at 37oC with 5% CO2 for 21 days. The conditioned culture media were collected and analyzed to probe for functional groups, as well as possible molecular variations associated with cell differentiation and metabolism. The method permitted to identify functional groups of specific molecules in the conditioned medium such as cholesterol, phosphatidylinositol, triglycerides, beta-subunit polypeptides, amide regions and hydrogen bonds of proteins, in addition to DNA expression. In the present study, FT-Raman spectroscopy exhibited limited resolution since different molecules can express similar or even the same stretching vibrations, a fact that makes analysis difficult. There were no variations in the readings between the samples studied. In conclusion, FT-Raman spectroscopy did not meet expectations under the conditions studied.

Resumo As células-tronco mesenquimais (MSCs) possuem grande potencial para aplicação em procedimentos terapêuticos ligados a terapia celular e engenharia de tecidos, considerando-se a plasticidade e capacidade de formação em diferentes tipos celulares por elas. Dada a abrangência no emprego das MSCs, há necessidade de se compreender melhor algumas propriedades relacionadas à diferenciação osteogênica, particularmente liga à biomateriais usados em engenharia de tecidos. Este projeto objetiva o desenvolvimento de uma metodologia de análise empregando-se a FT-Raman para identificação e quantificação de componentes bioquímicos presentes em meios de cultura condicionados por MSCs, com ou sem indução à diferenciação osteogênica. Todos os experimentos foram realizados entre as passagens 3 e 5. Para essas análises, as MSCs foram cultivadas sobre arcabouços de polímeros biorreabsorvíveis de poli (hidroxibutirato-co-hidroxivalerato) (PHBV) e o poli (ε-caprolactona) (PCL). As MSCs (GIBCO®) foram inoculadas nos polímeros puros e na mistura 75:25 de PHBV / PCL (amostras densas e porosas). As células foram mantidas em DMEM (com baixa glicose) contendo GlutaMAX® e 10% de SFB a 37oC com 5% de CO2 por 21 dias. A própria placa foi usada como controle. Os meios de cultura condicionados foram coletados e analisadas em FT-Raman para sondagem de grupos funcionais, bem como possíveis variações moleculares associadas com a diferenciação e metabolismo celular. Foi possível discernir grupos funcionais de moléculas específicas no meio condicionado, como colesterol, fosfatidilinositol, triglicerídeos, forma Beta de polipeptídeos, regiões de amida e ligações de hidrogênio de proteínas, além da expressão de DNA. Na presente avaliação, a FT-Raman apresentou como uma técnica de resolução limitada, uma vez que modos vibracionais de estiramento próximos ou mesmo iguais podem ser expressos por moléculas diferente, dificultando a análise. Não houve variações nas leituras entre as amostras estudadas, concluindo-se que a FT-Raman não atendeu às expectativas nas condições estudadas.

Effects of Zuogui Pill on osteoclast activity and expression of miR-133b-3p/RhoA in postmenopausal osteoporosis rats / 国际中医中药杂志

Objective:To study the effects of Zuogui Pills on the expressions of miR-133b-3p and RhoA in osteoclasts of postmenopausal osteoporosis rats; To discuss its potential mechanism.Methods:SD female rats were randomly divided into normal group, model group, sham-operation group, and Zuogui Pills group using a random number table method, with 6 rats in each group. The model group and Zuogui Pills group were treated with oophorectomy to construct a rat model of osteoporosis. Zuogui Pills group was orally administered with Zuogui Pills decoction at a concentration of 10 g/kg for 12 consecutive weeks. Colorimetric method was used to measure the serum calcium and phosphorus levels of rats, and ELISA method was used to detect ALP levels. Bone density meter was used to measure the bone density of the femurs of rats in each group. The osteoclast of each group were cultured, and the expressions of RANKL and RUNX2 protein were detected by Western blot. MiRNA sequencing and differential expression analysis were performed on bone tissues of rats. Osteoclasts were treated with miR-133b-3p mimic and its negative control. The cell proliferation activity of osteoclasts was detected by cell counting kit-8 (CCK-8). The osteoclast differentiation activity was detected by the tartrate-resistant acid phosphatase staining. The dual-luciferase reporter assay was used to detect the relationship between miR-133b-3p and RhoA. The "rescue" experiment of miR-133b-3p mimic and RhoA co-expression were used to study the molecular regulatory mechanism of Zuogui Pills on osteoclast activity.Results:Compared with the model group, the bone mineral density of Zuogui Pills group significantly increased ( P<0.05, P<0.01), the levels of calcium and phosphorus in serum increased, the level of alkaline phosphatase ALP decreased ( P<0.05), the expression of RANKL protein decreased, and the expression of RUNX2 protein increased. Sequencing results showed that rno-miR-133b-3p was down-regulated in osteoclasts of postmenopausa osteoporosis rats treated with Zuogui Pills with the maximum difference ( P<0.01). Q-PCR results showed that the expression of miR-133b-3p in osteoclasts of Zuogui Pills group was significantly lower than that of the model group. The upregulation of miR-133b-3p could significantly promote the cell proliferation and differentiation of osteoclasts. RhoA overexpression could reverse the excessive proliferation and differentiation of osteoclasts caused by miR-133b-3p overexpression. Conclusions:RhoA is the target gene regulated by miR-133b-3p. Zuogui Pills can inhibit the activity of osteoclasts by regulating miR-133b-3p/RhoA axis, relieving the symptoms of osteoporosis.

Effects of Cyclic Strain on Neural Differentiation of Rat Bone Marrow-Derived Mesenchymal Stem Cells / 医用生物力学

Objective To study the mechanical effects of cyclic strain on neural differentiation of rat bone marrow mesenchymal stem cells (rBMSCs). Methods The rBMSCs were subjected to cyclic strain for 24 hours andthen cultured for 5 days. The expression of neural markers and the phosphorylation of relative signaling pathway proteins were evaluated. The stress distribution on cell surface was analyzed by finite element method. The differentially expressed genes induced by strain were identified by RNA sequencing analysis. Results The 0. 5 Hz strain with 5% magnitude could significantly induce higher expression of neural markers and elevated phosphorylation level of extracellular-signal-regulated kinase (ERK), protein kinase B (AKT) and mammalian target of rapamycin ( mTOR). KEGG pathway analysis showed that the focal adhesion and ECM-receptor interaction were significantly enriched under cyclic strain. Conclusions Cyclic strain could change the interaction of cells with the extracellular matrix ( ECM) and enhance the AKT/ mTOR and ERK pathway, finally promote rBMSC neural differentiation. Knowledge about the impact of mechanical stimulation on BMSC neural differentiation is expected to improve the efficiency of stem cell differentiation, shed light on device design for tissue engineering, and promote clinical application of mesenchymal stem cells in neural issue repair and regeneration.

Cloning, identification and functional analysis of the goat transcription factor c-fos / 生物工程学报

C-fos is a transcription factor that plays an important role in cell proliferation, differentiation and tumor formation. The aim of this study was to clone the goat c-fos gene, clarify its biological characteristics, and further reveal its regulatory role in the differentiation of goat subcutaneous adipocytes. We cloned the c-fos gene from subcutaneous adipose tissue of Jianzhou big-eared goats by reverse transcription-polymerase chain reaction (RT-PCR) and analyzed its biological characteristics. Using real-time quantitative PCR (qPCR), we detected the expression of c-fos gene in the heart, liver, spleen, lung, kidney, subcutaneous fat, longissimus dorsi and subcutaneous adipocytes of goat upon induced differentiation for 0 h to 120 h. The goat overexpression vector pEGFP-c-fos was constructed and transfected into the subcutaneous preadipocytes for induced differentiation. The morphological changes of lipid droplet accumulation were observed by oil red O staining and bodipy staining. Furthermore, qPCR was used to test the relative mRNA level of the c-fos overexpression on adipogenic differentiation marker genes. The results showed that the cloned goat c-fos gene was 1 477 bp in length, in which the coding sequence was 1 143 bp, encoding a protein of 380 amino acids. Protein structure analysis showed that goat FOS protein has a basic leucine zipper structure, and subcellular localization prediction suggested that it was mainly distributed in the nucleus. The relative expression level of c-fos was higher in the subcutaneous adipose tissue of goats (P < 0.05), and the expression level of c-fos was significantly increased upon induced differentiation of subcutaneous preadipocyte for 48 h (P < 0.01). Overexpression of c-fos significantly inhibited the lipid droplets formation in goat subcutaneous adipocytes, significantly decreased the relative expression levels of the AP2 and C/EBPβ lipogenic marker genes (P < 0.01). Moreover, AP2 and C/EBPβ promoter are predicted to have multiple binding sites. In conclusion, the results indicated that c-fos gene was a negative regulatory factor of subcutaneous adipocyte differentiation in goats, and it might regulate the expression of AP2 and C/EBPβ gene expression.

DNA methylation analysis identifies key transcription factors involved in mesenchymal stem cell osteogenic differentiation

BACKGROUND: Knowledge about regulating transcription factors (TFs) for osteoblastogenesis from mesenchymal stem cells (MSCs) is limited. Therefore, we investigated the relationship between genomic regions subject to DNA-methylation changes during osteoblastogenesis and the TFs known to directly interact with these regulatory regions. RESULTS: The genome-wide DNA-methylation signature of MSCs differentiated to osteoblasts and adipocytes was determined using the Illumina HumanMethylation450 BeadChip array. During adipogenesis no CpGs passed our test for significant methylation changes. Oppositely, during osteoblastogenesis we identified 2462 differently significantly methylated CpGs (adj. p < 0.05). These resided outside of CpGs islands and were significantly enriched in enhancer regions. We confirmed the correlation between DNA-methylation and gene expression. Accordingly, we developed a bioinformatic tool to analyse differentially methylated regions and the TFs interacting with them. By overlaying our osteoblastogenesis differentially methylated regions with ENCODE TF ChIP-seq data we obtained a set of candidate TFs associated to DNA-methylation changes. Among them, ZEB1 TF was highly related with DNA-methylation. Using RNA interference, we confirmed that ZEB1, and ZEB2, played a key role in adipogenesis and osteoblastogenesis processes. For clinical relevance, ZEB1 mRNA expression in human bone samples was evaluated. This expression positively correlated with weight, body mass index, and PPARγ expression. CONCLUSIONS: In this work we describe an osteoblastogenesis-associated DNA-methylation profile and, using these data, validate a novel computational tool to identify key TFs associated to age-related disease processes. By means of this tool we identified and confirmed ZEB TFs as mediators involved in the MSCs differentiation to osteoblasts and adipocytes, and obesity-related bone adiposity.

Culture of rat mesenchymal stem cells on PHBV-PCL scaffolds: analysis of conditioned culture medium by FT Raman spectroscopy / Cultivo de células-tronco mesenquimais de rato sobre arcabouços de PHBV-PCL: análise do meio de cultura condicionado por espectroscopia de FT-Raman

Mesenchymal stem cells (MSCs) have great potential for application in cell therapy and tissue engineering procedures because of their plasticity and capacity to differentiate into different cell types. Given the widespread use of MSCs, it is necessary to better understand some properties related to osteogenic differentiation, particularly those linked to biomaterials used in tissue engineering. The aim of this study was to develop an analysis method using FT-Raman spectroscopy for the identification and quantification of biochemical components present in conditioned culture media derived from MSCs with or without induction of osteogenic differentiation. All experiments were performed between passages 3 and 5. For this analysis, MSCs were cultured on scaffolds composed of bioresorbable poly(hydroxybutyrate co-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL) polymers. MSCs (GIBCO®) were inoculated onto the pure polymers and 75:25 PHBV/PCL blend (dense and porous samples). The plate itself was used as control. The cells were maintained in DMEM (with low glucose) containing GlutaMAX® and 10% FBS at 37ºC with 5% CO2 for 21 days. The conditioned culture media were collected and analyzed to probe for functional groups, as well as possible molecular variations associated with cell differentiation and metabolism. The method permitted to identify functional groups of specific molecules in the conditioned medium such as cholesterol, phosphatidylinositol, triglycerides, beta-subunit polypeptides, amide regions and hydrogen bonds of proteins, in addition to DNA expression. In the present study, FT-Raman spectroscopy exhibited limited resolution since different molecules can express similar or even the same stretching vibrations, a fact that makes analysis difficult. There were no variations in the readings between the samples studied. In conclusion, FT-Raman spectroscopy did not meet expectations under the conditions studied.

As células-tronco mesenquimais (MSCs) possuem grande potencial para aplicação em procedimentos terapêuticos ligados a terapia celular e engenharia de tecidos, considerando-se a plasticidade e capacidade de formação em diferentes tipos celulares por elas. Dada a abrangência no emprego das MSCs, há necessidade de se compreender melhor algumas propriedades relacionadas à diferenciação osteogênica, particularmente liga à biomateriais usados em engenharia de tecidos. Este projeto objetiva o desenvolvimento de uma metodologia de análise empregando-se a FT-Raman para identificação e quantificação de componentes bioquímicos presentes em meios de cultura condicionados por MSCs, com ou sem indução à diferenciação osteogênica. Todos os experimentos foram realizados entre as passagens 3 e 5. Para essas análises, as MSCs foram cultivadas sobre arcabouços de polímeros biorreabsorvíveis de poli (hidroxibutirato-co-hidroxivalerato) (PHBV) e o poli (ε-caprolactona) (PCL). As MSCs (GIBCO®) foram inoculadas nos polímeros puros e na mistura 75:25 de PHBV / PCL (amostras densas e porosas). As células foram mantidas em DMEM (com baixa glicose) contendo GlutaMAX® e 10% de SFB a 37oC com 5% de CO2 por 21 dias. A própria placa foi usada como controle. Os meios de cultura condicionados foram coletados e analisadas em FT-Raman para sondagem de grupos funcionais, bem como possíveis variações moleculares associadas com a diferenciação e metabolismo celular. Foi possível discernir grupos funcionais de moléculas específicas no meio condicionado, como colesterol, fosfatidilinositol, triglicerídeos, forma Beta de polipeptídeos, regiões de amida e ligações de hidrogênio de proteínas, além da expressão de DNA. Na presente avaliação, a FT-Raman apresentou como uma técnica de resolução limitada, uma vez que modos vibracionais de estiramento próximos ou mesmo iguais podem ser expressos por moléculas diferente, dificultando a [...].

Culture of rat mesenchymal stem cells on PHBV-PCL scaffolds: analysis of conditioned culture medium by FT-Raman spectroscopy

Abstract Mesenchymal stem cells (MSCs) have great potential for application in cell therapy and tissue engineering procedures because of their plasticity and capacity to differentiate into different cell types. Given the widespread use of MSCs, it is necessary to better understand some properties related to osteogenic differentiation, particularly those linked to biomaterials used in tissue engineering. The aim of this study was to develop an analysis method using FT-Raman spectroscopy for the identification and quantification of biochemical components present in conditioned culture media derived from MSCs with or without induction of osteogenic differentiation. All experiments were performed between passages 3 and 5. For this analysis, MSCs were cultured on scaffolds composed of bioresorbable poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and poly(-caprolactone) (PCL) polymers. MSCs (GIBCO®) were inoculated onto the pure polymers and 75:25 PHBV/PCL blend (dense and porous samples). The plate itself was used as control. The cells were maintained in DMEM (with low glucose) containing GlutaMAX® and 10% FBS at 37oC with 5% CO2 for 21 days. The conditioned culture media were collected and analyzed to probe for functional groups, as well as possible molecular variations associated with cell differentiation and metabolism. The method permitted to identify functional groups of specific molecules in the conditioned medium such as cholesterol, phosphatidylinositol, triglycerides, beta-subunit polypeptides, amide regions and hydrogen bonds of proteins, in addition to DNA expression. In the present study, FT-Raman spectroscopy exhibited limited resolution since different molecules can express similar or even the same stretching vibrations, a fact that makes analysis difficult. There were no variations in the readings between the samples studied. In conclusion, FT-Raman spectroscopy did not meet expectations under the conditions studied.

Resumo As células-tronco mesenquimais (MSCs) possuem grande potencial para aplicação em procedimentos terapêuticos ligados a terapia celular e engenharia de tecidos, considerando-se a plasticidade e capacidade de formação em diferentes tipos celulares por elas. Dada a abrangência no emprego das MSCs, há necessidade de se compreender melhor algumas propriedades relacionadas à diferenciação osteogênica, particularmente liga à biomateriais usados em engenharia de tecidos. Este projeto objetiva o desenvolvimento de uma metodologia de análise empregando-se a FT-Raman para identificação e quantificação de componentes bioquímicos presentes em meios de cultura condicionados por MSCs, com ou sem indução à diferenciação osteogênica. Todos os experimentos foram realizados entre as passagens 3 e 5. Para essas análises, as MSCs foram cultivadas sobre arcabouços de polímeros biorreabsorvíveis de poli (hidroxibutirato-co-hidroxivalerato) (PHBV) e o poli (-caprolactona) (PCL). As MSCs (GIBCO®) foram inoculadas nos polímeros puros e na mistura 75:25 de PHBV / PCL (amostras densas e porosas). As células foram mantidas em DMEM (com baixa glicose) contendo GlutaMAX® e 10% de SFB a 37oC com 5% de CO2 por 21 dias. A própria placa foi usada como controle. Os meios de cultura condicionados foram coletados e analisadas em FT-Raman para sondagem de grupos funcionais, bem como possíveis variações moleculares associadas com a diferenciação e metabolismo celular. Foi possível discernir grupos funcionais de moléculas específicas no meio condicionado, como colesterol, fosfatidilinositol, triglicerídeos, forma Beta de polipeptídeos, regiões de amida e ligações de hidrogênio de proteínas, além da expressão de DNA. Na presente avaliação, a FT-Raman apresentou como uma técnica de resolução limitada, uma vez que modos vibracionais de estiramento próximos ou mesmo iguais podem ser expressos por moléculas diferente, dificultando a análise. Não houve variações nas leituras entre as amostras estudadas, concluindo-se que a FT-Raman não atendeu às expectativas nas condições estudadas.

Micro/nano hierarchical structure of titanium surface promotes MC3T3-E1 adhesion proliferation and drug loading potential evaluation / 口腔疾病防治

Objective @# To investigate the effect of micro/nano hierarchical structures on the adhesion and proliferation of MC3T3-E1 cells, evaluate the drug delivery potential of titanium surfaces, and provide a reference for the modification of selected areas of titanium surfaces to enhance drug delivery and slow drug release. @*Methods @# Pure titanium samples (10 mm in diameter and 2.5 mm in thickness) were randomly divided into a polished group (T), anodized group (TO), and micro/nano hierarchical structure group (FTO) according to the surface treatment of the titanium. The T group was polished, the TO group was treated with anodic oxidation technology, and the FTO group was treated by femtosecond laser etching combined with anodic oxidation technology. The three surface morphologies were observed by scanning electron microscopy (SEM), the wettability of the surface was measured by the contact angle, and the surface chemical composition was analyzed by X-ray energy dispersive spectroscopy (EDS). The depth of the FTO structure and the surface roughness were measured by confocal laser scanning microscopy (CLSM). MC3T3-E1 cell adhesion proliferation and differentiation on the surface of each group of samples was assessed by immunofluorescence staining, CCK-8, and semiquantitative analysis of Alizarin staining. A freeze-drying method was applied to load recombinant human bone morphogenetic protein-2 (rhBMP-2), and an enzyme-linked immunosorbent assay (ELISA) was used to assess the drug-loading potential of different surface structures. @* Results@#SEM revealed that the surface of T group titanium plates showed uniform polishing marks in the same direction. The surface of the TO group was a nanoscale honeycomb-like titanium dioxide (TiO2) nanotube structure, and the FTO group formed a regular and ordered micro/nano layered structure. The contact angle of the FTO group was the smallest at 32° ± 1.7°. Its wettability was the best. The average depth of the first-level structure circular pores was 93.6 μm, and the roughness was 1.5-2 μm. The TO and FTO groups contained a high percentage of oxygen, suggesting TiO2 nanotube formation. The FTO group had the most significant surface cell proliferation (P<0.001) and the largest cell adhesion surface area (P<0.05). rhBMP-2 was slowly released for 14 d after loading in the FTO group and promoted extracellular matrix mineralization (P<0.001). @*Conclusion @#Titanium surface microprepared hierarchical structure has the effect of promoting MC3T3-E1 cell adhesion, proliferation, and osteogenic differentiation with drug loading potential, which is a new method of titanium surface treatment.

Teriparatide regulates osteoblast differentiation in high-glucose microenvironment through the cAMP/PKA/CREB signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate the effect of teriparatide on the differentiation of MC3T3-E1 cells in high-glucose microenvironment and explore the possible mechanism.@*METHODS@#MC3T3-E1 cells cultured in normal glucose or high-glucose (25 mmol/L) medium were treated with 10 nmol/L teriparatide with or without co-treatment with H-89 (a PKA inhibitor). CCK-8 assay was used to detect the changes in cell proliferation, and cAMP content in the cells was determined with ELISA. Alkaline phosphatase (ALP) activity and mineralized nodules in the cells were detected using ALP kit and Alizarin red staining, respectively. The changes in cell morphology were detected by cytoskeleton staining. Real-time PCR was used to detect the mRNA expressions of PKA, CREB, RUNX2 and Osx in the treated cells.@*RESULTS@#The treatments did not result in significant changes in proliferation of MC3T3-E1 cells (P > 0.05). Compared with the cells in routine culture, the cells treated with teriparatide showed significantly increased cAMP levels (P < 0.05) with enhanced ALP activity and increased area of mineralized nodules (P < 0.05). Teriparatide treatment also resulted in more distinct visualization of the cytoskeleton in the cells and obviously up-regulated the mRNA expressions of PKA, CREB, RUNX2 and Osx (P < 0.05). The opposite changes were observed in cells cultured in high glucose. In cells exposed to high glucose, treatment with teriparatide significantly increased cAMP levels (P < 0.05), ALP activity and the area of mineralized nodules (P < 0.05) and enhanced the clarity of the cytoskeleton and mRNA expressions of PKA, CREB, RUNX2 and Osx; the effects of teriparatide was strongly antagonized by co-treatment with H-89 (P < 0.05).@*CONCLUSION@#Teriparatide can promote osteoblast differentiation of MC3T3-E1 cells in high-glucose microenvironment possibly by activating the cAMP/PKA/CREB signaling pathway.

Organoides: fundamentos, presente y futuro / Organoids: fundamentals, present and future

RESUMEN Los organoides son estructuras miniaturizadas, generadas principalmente a partir de células madre pluripotentes inducidas, que se cultivan en el laboratorio conservando sus características innatas o adquiridas. Tienen el potencial de reproducir procesos de desarrollo biológico, modelar procesos patológicos que permitirán el descubrimiento de nuevos fármacos y propicien la medicina regenerativa. Sin embargo, estas experiencias requieren perfeccionamiento constante porque pueden haberse realizado variaciones en la constitución de estos órganos. Por ello, el presente artículo tiene como objetivo revisar la información actualizada sobre organoides y sus procesos experimentales básicos y recientes, empezando por la gastrulación, para tratar de imitar, en lo posible, la formación de las tres capas: ectodermo, mesodermo y endodermo, incluyendo los factores que intervienen en la inducción, diferenciación y maduración en la generación de estos organoides. Asimismo, el diseño y preparación de medios de cultivo altamente especializados que permitan obtener el órgano seleccionado con la mayor precisión y seguridad. Se realizó una búsqueda de artículos originales y de revisión publicados en PubMed, Nature y Science. Los artículos se seleccionaron por sus resúmenes y por su texto completo. Las conclusiones de este articulo destacan las ventajas futuras en el uso y aplicaciones de los organoides.

ABSTRACT Organoids are tiny structures, mainly generated from induced pluripotent stem cells, which are cultured in the laboratory while retaining their innate or acquired characteristics. They have the potential to reproduce biological development processes, model pathological processes that will enable the discovery of new drugs and promote regenerative medicine. However, these processes require constant improvement because variations may have occurred in the constitution of the organs. Therefore, this article aims to review updated information on organoids and their basic and recent experimental processes, starting with gastrulation, in an attempt to mimic, as much as possible, the formation of the three layers: ectoderm, mesoderm and endoderm; as well as the information regarding the factors involved in the induction, differentiation and maturation during the generation of organoids. Likewise, the design and preparation of highly specialized culture media that allow obtaining the selected organ with the highest precision and safety. We searched for original and review articles published in PubMed, Nature and Science. Articles were selected for their abstracts and full text. The conclusions of this article highlight the future advantages in the use and applications of organoids.

Studies on the differentiation of human urine derived stem cells into nucleus pulposus-like cells induced by human nucleus pulposus cell exosomes / 中华骨科杂志

Objective:To investigate the effects of exosomes of human nucleus pulposus cells (NPCs) on the differentiation of urine derived stem cells (USCs) into nucleus pulposus-like cells.Methods:USCs and NPCs were isolated and cultured in vitro. The exosomes of NPCs were extracted and detected by Western-blot. USCs cytoplasm was transfected with GFP lentivirus, while nucleus was transfected with DAPI dye. The NPCs exosomes were transfected with PKH26 dye. After co-incubation for 12 h, USCs and NPCs exosomes were observed by macroscopy. USCs differentiation was induced by NPCs exosomes and non-contact co-culture methods. The relative expression of marker gene mRNA of nucleus pulposus cells in each group and the absorbance at 450 nm wavelength were detected.Results:The isolated USCs had the ability to differentiate into osteocytes, adipocytes and chondrocytes with high expression of marker CD29 (99.57%), CD44 (97.46%) and CD73 (97.71%) and with low expression of negative proteins CD31 (0.59%) and CD45 (0.19%). The isolated NPCs highly expressed nuclear pulposus cell marker COL2A1, ACAN and SOX-9. The exosomes extracted from NPCs showed high expression of exosome marker CD63, CD81 and Tsg101. After 12 h co-incubation, NPCs exosomes fused with USCs membrane and appeared in the cytoplasm of USCs. At 3, 5 and 7 days of co-culture, the absorbance value of USCs cells in exosome group (0.44±0.004, 0.76±0.004, 0.82±0.006) was higher than that in co-culture group (0.39±0.022, 0.63±0.035, 0.69±0.012) ( P<0.05). The mRNA relative expression of USCs nucleus pulposus marker genes ACAN (1.80±0.31, 3.50±0.21, 5.35±0.31, 7.46±0.12), COL2A1 (1.43±0.15, 4.33±0.23, 6.89±0.22, 8.11±0.31), SOX-9 (2.21±0.13, 3.13±0.11, 3.96± 0.14, 4.52±0.26) and HIF-1α (1.45±0.16, 2.14±0.21, 4.31±0.41, 4.01±0.25) in exosomes group were significantly higher than those in the control group ( P<0.05) at the 3rd, 7th, 14th and 21st days. The mRNA relative expression of USCs nucleus pulposus marker genes ACAN (5.69±0.21, 6.69±0.13), COL2A1 (6.33±0.17, 7.89±0.15), SOX-9 (4.19±0.29, 4.38±0.12), HIF-1α (4.49±0.32, 4.96±0.26) in exosomes group were significantly higher than those ACAN (3.69±0.35, 5.13±0.23), COL2A1 (3.40±0.16, 6.79±0.19), SOX-9 (2.26±0.32, 3.69±0.26), HIF-1α (2.39±0.11, 3.96±0.13) in non-contact co-culture group ( P<0.05) at the 14th and 21st days. Conclusion:Human nucleus pulposus exosomes could induce differentiation of human USCs into nucleus pulposus-like cells in vitro. Compared with non-contact co-culture, exosomes have higher induction efficiency and can better maintain the proliferation activity of nucleus pulposus-like cells

Study on the differentiation of rat bone marrow mesenchymal stem cells into islet-like cell masses under co-culture conditions / 中华胰腺病杂志

Objective:To examine the possibility of the differentiation into islet-like cell clusters from the co-culture system of bone marrow mesenchymal stem cells (BMSCs) and islet cells.Methods:Rat BMSCs from the femur and tibia of Wistar rats were isolated and purified taken under aseptic conditions; the surface markers CD 44 and CD 90 expressions of BMSCs were detected by flow cytometry; and alizarin red staining and oil red O staining were used to identify the cells induced in the osteogenic direction and adipogenic direction, respectively. Rat islet cells from the pancreas of Wistar rats were isolated and purified; and dithiazone staining was performed for validation. The basal insulin level of the culture was detected by ELISA method. 5.6mmol/L (low glucose) and 25.0 mmol/L (high glucosa) glucose were added to the culture, respectively, and insulin release was detected by ELISA. 5-generation BMSCs and islet cells were collected and divided randomly into stem cell culture alone group (stem cell group), stem cell-islet co-culture group (co-culture group), and islet culture alone group (islet group). The morphological changes of BMSCs during co-culture were observed using an inverted phase contrast microscope; basal insulin secretion and insulin secretion stimulated by low and high glucose were tested by ELISA. Insulin protein expression in induced islet-like cell masses in co-culture group were detected by immunocytochemical staining. The ultrastructure of islet-like cells was observed by using transmission electron microscopy. Results:The positive rates of CD 44 and CD 90 were 99.48% and 99.50%, respectively; BMSCs were induced the formation of multiple calcium nodules outside the differentiation cells in the osteogenic direction, and many lipid droplets in the cytoplasm of differentiated cells in the adipogenic direction. Dithiazone staining showed that β cells in pancreatic islet were brown red and about 450 islets could be obtained per pancreas with a mean purity up to 80%. The insulin release in the low sugar group and the high sugar group were (7.105±1.551) mIU/ml and (20.231±1.592) mIU/ml, respectively, with a statistically significant difference ( P<0.05). It can be seen that local stem cells began to gather and grow upward into small clumps in the budding manner until finally forming a spherical islet-like cell cluster structure after 7 days of culture in the co-culture group. The basal insulin secretion in the stem cell group was <0.5 mIU/L. In the islet group, insulin secretion peaked on the 5th day and then gradually decreased to about 20% of the highest value on the 13th day. The insulin level of the co-culture group peaked on the 5th day, and the 13th day remained at about 40% of the peak level. There were statistically significant differences on basal insulin secretion on the 8th, 10th and 13th day between islet group and co-culture group (all P value >0.05). There was no statistically significant difference between the insulin release by islet in islet group under the stimulation of low and high sugar and that by islet-like cell cluster in co-culture group. There were a large number of brownish-yellow granules in the islet-like cell clusters after the co-culture for 14 days; and there were more secretory granules and coarse endoplasmic reticulum in the ultrastructure, showing more active protein secretion functions. Conclusions:The co-culture system of BMSCs and islet cells could induce BMSCs into differentiating into islet-like cell clusters, which can express insulin protein and had relatively mature function of insulin secretion.

Promoting effect of nicotinamide on generation of neural crest stem cells derived from human embryonic stem cells / 中华实验眼科杂志

Objective:To investigate the role of nicotinamide (NIC) in the differentiation of neural crest cells from human embryonic stem cells (hESCs), and lay the foundation for the induction of hESC-derived corneal endothelial cells.Methods:hESCs line H1 cultured for 5-7 days was used for induction.According to the different components of the neural crest induction medium, cells were assigned into different groups for 7-days induction, including group treated without NIC cultured in induction medium only, group treated with NIC cultured in induction medium containing 10 mmol/L NIC, NIC+ resveratrol (Res) group cultured in induction medium containing 10 mmol/L NIC and 10 μmol/L Res and Sirtinol group cultured in induction medium containing 10 μmol/L Sirtinol.Res and Sirtinol were used as SIRT1 activity agonist and inhibitor, respectively.The relative mRNA expression levels of hESCs and neural crest cell markers were detected by real-time fluorescence quantitative PCR at 1, 3, 5 and 7 days during the induction.The expression of neural crest cells markers after 7 days of induction was assayed by immunofluorescence staining.The induction efficiency of NIC and the effect of SIRT1 regulation on human natural killer 1 (HNK-1) positive cells expression were evaluated through flow cytometry analysis of percentages of nerve growth factor receptor (P75) and HNK-1 + cells. Results:Compared with the group treated without NIC, the mRNA expressions of totipotent genes octamer transcription factor 4 (OCT4) and homeodomain proteins (NANOG) were significantly decreased, and the mRNA expression levels of neural crest cell markers P75, HNK-1, SRY-related HMG box (SOX) 9 and SOX10 were significantly increased in the group treated with NIC after 5 days of induction (all at P<0.05). In the group treated without NIC, P75 was weakly expressed, and HNK-1 was sporadically expressed, and transcription factor AP-2β (AP-2β) and paired-like homeodomain transcription factor 2 (PITX2) were not detected.In the group treated with NIC, P75, HNK-1, AP-2β and PITX2 were strongly expressed.The proportion of P75 + HNK-1 + cells and P75 + cells were both significantly higher in the group treated with NIC than without NIC ( t=8.481, P=0.001; t=2.987, P=0.041). The percentage of HNK-1 + cells in groups treated without and with NIC, NIC+ Res group and Sirtinol group were (34.267±12.522)%, (89.633±1.358)%, (64.667±6.429)% and (86.300±3.460)%, respectively, with a statistically significant overall difference ( F=36.799, P<0.001). The proportion of HNK-1 + cells in NIC+ Res group was significantly lower than that in the groups treated with NIC and Sirtinol (all at P<0.05). Conclusions:NIC promotes the differentiation of hESCs-derived neural crest cells by inhibiting the activity of SIRT1 to enhance the expression of HNK-1.NIC treatment may provide a new strategy for source of seed cells in the treatment of neural crest cell-related diseases, such as corneal endothelial transplantation.

Pressure-induced differentiation of rabbit retinal stem cells into retinal ganglion cells / 中华实验眼科杂志

Objective:To investigate the effect of pressure on the differentiation of rabbit retinal stem cells (RSCs) co-cultured with retinal ganglion cells (RGCs).Methods:SPF grade New Zealand rabbits on the day 22 of gestation were selected, and embryos were removed to obtain retinal ciliary margin pigment epithelial tissue and culture primary RSCs.Six SPF grade newborn New Zealand rabbits were selected, and retinal neuroepithelial layer tissues were isolated to culture primary RGCs.Rabbit RSCs cultured in vitro were identified by immunofluorescence staining of nestin antibody, bromodeoxyuridine (BrdU) cell proliferation assay kit, RSCs spontaneously differentiated cells immunofluorescence detection and flow cytometry.RGCs were identified through immunofluorescence staining of Brn3b antibody and Thy1.1 antibody.A co-culture system of RGCs and RSCs cultured in the upper and lower layers of a transwelll plate respectively was constructed.The mRNA and protein expression levels of nestin and Thy1.1 in RSCs and differentiated cells under pressures of 0, 20, 40, 60, 80 mmHg (1 mmHg=0.133 kPa) were detected by real-time fluorescence quantitative PCR and Western blot.The feeding and use of laboratory animals were in accordance with the Regulations on the Administration of Laboratory Animals promulgated by the State Science and Technology Commission.The study protocol was approved by the Ethics Committee of Yunnan University Affiliated Hospital (No.KPRC-IACUC17008). Results:RSCs cultured in vitro were nestin-positive.The percentage of BrdU-positive isolated RSCs was (92.26±3.28)%.Some cells differentiated from RSCs were Brn3b-positive, accounting for (13.00±3.06)%, and some were GS-positive, accounting for (31.60±3.67)%.RGCs cultured in vitro were Brn3b- and Thy1.1-positive.There were statistically significant differences in the relative mRNA and protein expressions of nestin and Thy1.1 between RSCs and differentiated cells under different pressures (mRNA: F=127.600, 137.400; both at P<0.01; protein: F=82.480, 158.700; both at P<0.001). The relative mRNA and protein expressions of nestin were significantly reduced in RSCs, and relative mRNA and protein expressions of Thy1.1 were significantly increased in differentiated cells at 20, 40, 60 and 80 mmHg in comparison with 0 mmHg (all at P<0.05). When the pressure was 40 mmHg, the relative mRNA and protein expressions of nestin were lowest in RSCs, and the relative mRNA and protein expressions of Thy1.1 in differentiated cells were highest. Conclusions:Within a certain range, pressure can promote the differentiation of RSCs co-cultured with RGCs into ganglion-like cells, and excessive pressure can inhibit the differentiation of RSCs.

The promotion of bone formation at the tendon-bone interface after ACL reconstruction with De-BMSCs transplantation and its mechanism / 中华骨科杂志

Objective:This study aimed to investigate the effect of differentiation osteogenic bone marrow mesenchymal stem cells (De-BMSCs) transplantation on the promotion of bone formation at the tendon-bone interface after anterior cruciate ligament reconstruction (ACLR), and further explored the molecular mechanism of the enhanced osteogenic effect of De-BMSCs.Methods:BMSCs from femur and tibia of New Zealand White rabbit were subjected to osteogenic induction and then cultured in no osteogenic factor medium; the obtained cell population was termed De-BMSCs. De-BMSCs were induced into osteo-, chondro-and adipo-differentiation in vitro to examine the characteristics of primitive stem cells. ACLR model with a semitendinosus tendon were performed in 48 adult rabbits, three groups were established: control group with alginate gel injectionat the tendon-bone interface, BMSCs group with the injection of alginate gel containing BMSCs, De-BMSCs group with the injection of alginate gel containing De-BMSCs. At 4 and 12 weeks after surgery, rabbits in each group were sacrificed to evaluate tendon-bone healing by histologic staining, micro-CT examination, and biomechanical test. During osteogenic differentiation of De-BMSCs, si-RNA of nuclear factor of activated T cells 2 (NFATc2) si-RNA of nuclear factor of activated T cells 1 (NFATc1) were used to verify the molecular mechanism of enhanced osteogenic effect of De-BMSCs.Results:De-BMSCs exhibited some properties similar to BMSCs including multiple differentiation potential and cell surface marker. At 4 weeks after surgery, the BV/TV value of the De-BMSCs group 0.36±0.01 was significantly higher than that of the control group 0.24±0.03 and BMSCs group 0.30±0.02 (all P<0.05), and the maximum load 40.34±1.19 N and stiffness 20.67±2.14 N/mm were significantly higher than those in the control group 14.88±2.74N, 8.67±2.19 N/mm and the BMSCs group 26.31±1.76 N, 13.81±2.14 N/mm (all P<0.05). At 12 weeks after surgery, the BV/TV value of the De-BMSCs transplantation group 0.47±0.02 was significantly higher than that of the control group 0.30±0.02 and the BMSCs group 0.35±0.03 (all P<0.05), and the maximum load 64.46±6.69 N and stiffness 25.18±3.11 N/mm were significantly higher than those in the control group 41.01±6.12 N, 11.59±2.54 N/mm and the BMSCs group 48.21±4.12 N, 15.89±2.94 N/mm (all P<0.05). During the osteogenic differentiation of De-BMSCs, the expressions of Nanog and NFATc1 were synergistically increased which promoted interaction of NFATc1 and Osterix ( P< 0.05), resulting in the increased expression of osteoblast marker genessuch as COL1A, OCN, OPN (all P< 0.05). Conclusion:De-BMSCs transplantation could promote bone formation at the tendon-bone interface after ACLR,Nanog/NFATc1/Osterix signaling pathway mediated the enhancement of the osteogenic differentiation effect of De-BMSCs.

Promoting effect of conditioned medium of human bone mesenchymal stem cells on proliferation, adhesion and neuronal differentiation of immortalized human Müller cell line / 中华实验眼科杂志

Objective:To explore the effects of conditioned medium of human bone marrow mesenchymal stem cells (BMSCs) on the proliferation, adhesion and differentiation of immortalized human Müller cell line (MIO-M1).Methods:The differentiation was induced in the third-passage BMSCs with osteogenic, chondrogenic and adipogenic medium and identified by alizarin red, alcian blue and oil red O staining, respectively.The expression levels of mesenchymal stem cell markers CD73, CD90 and CD105 and hematopoietic cell markers CD34, CD45 and human leukocyte antigen-DR (HLA-DR) were assayed by flow cytometry.The expressions levels of Müller cell markers SOX9, glutamine synthetase (GS), vimentin and cellular retinaldehyde-binding protein (CRALBP), retinal stem cell markers SOX2, nestin and CHX10, and cell proliferation marker cyclin D3 (CCND3) in MIO-M1 cells were detected by immunofluorescence staining.The MIO-M1 cells were divided into standard medium group, 293T conditioned medium group, and BMSC conditioned medium group and were incubated in the medium according to grouping.The cellular area, circularity, elongation factor and perimeter were analyzed quantitatively.The cell cycle was detected by flow cytometry, and the cell proliferation was determined by neurospora experiment and 5-ethynyl-2'-deoxyuridine (EdU) staining.The expression of vascular cell adhesion molecule 1 (VCAM-1) at protein and mRNA levels in the culture supernatant was detected by enzyme linked immunosorbent assay (ELISA) and quantitative real-time PCR (qRT-PCR), respectively.The expression of retinal neuron markers protein kinase C (PKCα), Rhodopsin, microtubule-associated protein 2 (MAP2) and β-tubulin (Tuj1) was detected by immunofluorescence staining and qRT-PCR.Results:CD73, CD90, CD105 showed an enhanced expression, and CD34, CD45 and HLA-DR showed weakened expression in the BMSCs.The BMSCs differentiated into osteoblasts, chondrocytes and adipocytes.Expression of SOX9, GS, vimentin and CRALBP, SOX2, CHX10, nestin and CCND3 was found in the MIO-M1 cells.Compared with standard medium group and 293T conditioned medium group, MIO-M1 cells cultured in BMSC conditioned medium group changed into an elongated spindle-shaped or multipolar morphology with reduced cell area, increased elongation index and decreased circularity, showing statistically significant differences among them ( F=6.973, 12.370, 6.311; all at P<0.01). There were increased neurospheres formed by MIO-M1 cells in BMSC conditioned medium group compared with standard medium group and 293T conditioned medium group at different time points ( Fgroup=134.300, P<0.001; Ftime=82.910, P<0.001). Compared with the standard medium group and 293T conditioned medium group, the EdU-positive rate and proliferation index of MIO-M1 cells in BMSC conditioned medium group were significantly increased, with statistically significant differences ( F=6.973, 74.110; all at P<0.05); the VCAM-1 protein expression in cell supernatant and the relative expression level of VCAM-1 mRNA in BMSC conditioed medium group were significantly increased ( F=13.720, 7.896; all at P<0.05); the mRNA expression levels of PKCα, Rhodopsin, Tuj1 and MAP2 were higher in MIO-M1 cells of BMSC conditioned medium group under the condition of differentiation ( F=14.490, 5.424, 14.330, 7.405; all at P<0.05). Conclusions:BMSCs conditioned medium can change the morphology of MIO-M1 cells and promote their proliferation, adhesion and differentiation into retinal neurons.

Role of glycolysis in Th17 cell differentiation in autoimmune diseases / 中华微生物学和免疫学杂志

T helper 17 (Th17) cells are closely associated with the pathogenesis of several autoimmune and inflammatory diseases, and selective suppression of Th17 cell production and pathogenicity is an effective strategy for the treatment of these diseases. There is growing evidence that cellular metabolism is associated with the development of autoimmune diseases and determines the differentiation and effector functions of Th17 cells, which undergoes a metabolic reorganization during differentiation from an oxidative phosphorylation-based catabolism to a glucose-based anabolic metabolism in the initial T cells. This paper focused on reviewing recent findings regarding the importance of metabolism in T cell differentiation and autoimmune diseases, especially in Th17 cells, and discussing the regulatory mechanisms of glycolysis in Th17 cell differentiation. This review summarized the regulation of metabolism on T-cell activation and differentiation, revealed metabolic targets with specific regulation on Th17 cells, and provided reference for finding potential therapeutic targets for Th17 cell-mediated autoimmune diseases.

Effects of repetitive magnetic stimulation on the growth and differentiation of SH-SY5Y cells / 中华物理医学与康复杂志

Objective:To explore the effect of repeated magnetic stimulation (rMS) on the growth and differentiation of SH-SY5Y human neuroblastoma cells.Methods:SH-SY5Y cells were subjected to rMS at 15%, 30% and 60% of the maximum output intensity at frequencies of 0.5Hz, 1Hz, 5Hz, 10Hz and 20Hz. They received either 800 or 1600 pulses per day for 4 days. Cell viability was detected using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay. Apoptosis was induced using 1-methyl-4-phenylpyridine ion (MPP + ) and all-trans retinoic acid was used to induce differentiation. The expression of neuron-specific nuclear proteins and the degree of cell differentiation were observed by immunohistochemistry. Results:0.5Hz rMS inhibited proliferation and 10Hz rMS promoted it. With 5Hz rMS significantly greater cell proliferation was observed at 15% and 30% of the maximum output intensity. The stimulatory effect of 1600 pulses per day was significantly greater than that of 800 pulses, especially at 10Hz. Apoptosis was inhibited at both 0.5Hz and 10Hz with 30% of the maximum output intensity. Meanwhile, both 0.5Hz and 10Hz rMS promoted differentiation of the SH-SY5Y cells into neurons.Conclusions:rMS at low frequency inhibits the proliferation of SH-SY5Y cells, but at higher frequency it promotes it. The effect strengthens with more pulses administered. rMS has a protective effect on MPP + -induced SH-SY5Y apoptosis, and it can promote the cells′ differentiation into neurons.

Effects of leptin on proliferation and differentiation of hypoxic rat retinal progenitor cells in vitro / 南方医科大学学报

OBJECTIVE@#To investigate the the effects of leptin on the proliferation, differentiation and PTEN expression of rat retinal progenitor cells (RPCs) cultured under hypoxic condition.@*METHODS@#SD rat RPCs were cultured in normoxic conditions or exposed to hypoxia in the presence of 0, 0.3, 1.0, 3.0, 10, and 30 nmol/L leptin for 12, 48 and 72 h, and the cell viability was assessed using cell counting kit 8 (CCK 8) assay. The RPCs in primary culture were divided into control group, hypoxia group, and hypoxia+leptin group, and after 48 h of culture, the cell medium was replaced with differentiation medium and the cells were further cultured for 6 days. Immunofluorescence staining was employed to detect the cells positive for β-tubulin III and GFAP, and Western blotting was used to examine the expression of PTEN at 48 h of cell culture.@*RESULTS@#The first generation of RPCs showed suspended growth in the medium with abundant and bright cellular plasma and formed mulberry like cell spheres after 2 days of culture. Treatment with low-dose leptin (below 3.0 nmol/L) for 48 h obviously improved the viability of RPCs cultured in hypoxia, while at high concentrations (above 10 nmol/L), leptin significantly suppressed the cell viability (P < 0.05). The cells treated with 3.0 nmol/L leptin for 48 h showed the highest viability (P < 0.05). After treatment with 3.0 nmol/L leptin for 48 h, the cells with hypoxic exposure showed similar GFAP and β-tubulin Ⅲ positivity with the control cells (P>0.05), but exhibited an obvious down-regulation of PTEN protein expression compared with the control cells (P < 0.05).@*CONCLUSION@#In rat RPCs with hypoxic exposure, treatment with low dose leptin can promote the cell proliferation and suppress cellular PTEN protein expression without causing significant effects on cell differentiation.