218 DONOR-MATCHED FUNCTIONAL AND MOLECULAR CHARACTERIZATION OF CANINE MESENCHYMAL STEM CELLS DERIVED FROM DIFFERENT ORIGINS

2012 ◽  
Vol 24 (1) ◽  
pp. 221
Author(s):  
S. A. Ock ◽  
G. H. Maeng ◽  
Y. M. Lee ◽  
T. H. Kim ◽  
B. M. Kumar ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Transcription Factors ◽  
Telomere Length ◽  
Telomerase Activity ◽  
Rt Pcr ◽  
Cytochemical Staining ◽  
Single Donor ◽  
Cell Capacity

Canine mesenchymal stem cells (cMSC) have been successfully isolated from several adult tissue sources. However, differences in the biological properties of MSC have been shown to be associated with donor variability. Further, the stem cell capacity of cMSC of various tissues isolated from a single donor is currently unclear. Therefore, this study investigated the functional and molecular characteristics of cMSC derived from bone marrow (cBM-MSC), adipose tissue (cA-MSC) and dermal skin (cDS-MSC) of a single donor. Three kinds of cMSC were isolated by following previously published protocols. AP activity was assessed with a chromogen kit (Abcam Inc., Cambridge, MA, USA). Expression of CD markers (CD45, 90 and 105) and stem cell transcription factors (Oct3/4, Nanog and Sox2) was analysed by immunocytochemical staining. All cells were induced into osteogenesis and adipogenesis by following protocols described earlier and confirmed by cytochemical staining and the detection of lineage specific genes by RT-PCR. Chromosomal stability was assessed by a method described earlier (Ock and Rho 2008 J. Vet. Med. Sci. 70, 1165–1172) and cell cycle status was determined by a flow cytometry. Telomere length was analysed by Telo TAGGG Telomere Length Assay kit (Roche, Mannheim, Germany) and telomerase activity was evaluated by semiquantitative nested RT-PCR. Statistical analysis was performed by ANOVA using SPSS 12.0 and significance was tested when P < 0.05. Expressions of AP activity and the transcription factors, such as Oct3/4, Nanog and Sox2 were absent in all cMSC. All 3 types of cMSC positively expressed the surface markers CD90 and 105 but not CD45. Exposure of all cell lines to osteogenic and adipogenic induction medium resulted in the calcium deposition evidenced by Alizarin red S staining and the accumulation of fat globules indicated by Oil red O staining, respectively. Differentiation was further confirmed by the detection of marker genes, such as Runx2 and Pparγ. However, the degree of osteogenic or adipogenic differentiation among the 3 kinds of cMSC was different and particularly, cA-MSC had enhanced cytochemical staining associated with expression of specific genes, Runx2 and Pparγ. Ploidy analysis showed that the diploid rate was high with over 90% in all cMSC and indicated no noticeable chromosomal abnormalities. Further, less than 52% of cells were found at G1 phase in all cMSC, with lowest percentage observed in cDS-MSC (33.3%). Regardless of varied tissue sources, cMSC from a single donor showed no differences in telomere lengths (∼18–19 kbp), but the telomerase activity was different with significantly higher levels found in cBM-MSC. In conclusion, the above results suggest that tissue specific cMSC derived from a single donor possess differences in stem cell capacity and support the consideration of tissue source before judging the suitability of cells for therapeutic applications. This work was supported by grant from Basic Science Research Program through NRF funded by the Ministry of Education, Science and Technology (2009-0064229).

scholarly journals Imetelstat Inhibits Telomerase and Prevents Propagation of ADAR1-Activated Myeloproliferative Neoplasm and Leukemia Stem Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 18-18
Author(s):  
Wenxue Ma ◽  
Larissa Balaian ◽  
Phoebe Mondala ◽  
Yudou He ◽  
Cayla Mason ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Telomere Length ◽  
Mouse Models ◽  
Telomerase Activity ◽  
Rna Seq ◽  
Publicly Traded ◽  
Cd34 Cells

BACKGROUND Clonal stem cell derived myeloproliferative neoplasms (MPNs) have a propensity to evolve to acute myeloid leukemia (AML). Deregulation of the innate immune deaminase associated with RNA1 (ADAR1) has been linked to malignant progression and therapeutic resistance. Increased expression of the stem cell gene, human telomerase reverse transcriptase (hTERT), has also been linked with malignant transformation. However, the combinatorial role of ADAR1 and hTERT in the evolution of MPN stem cells to therapy resistant acute myeloid leukemia stem cells (LSCs) and the capacity of a telomerase inhibitor, imetelstat, to prevent survival and self-renewal of pre-LSC and LSC had not been established. Recent clinical trials show early signs of efficacy of imetelstat in treatment of myelofibrosis (MF). However, its role in selectively inhibiting pre-LSC transformation to self-renewing LSC has not been elucidated. Here we show that targeting telomerase activity prevents pre-LSC and LSC maintenance both in vitro and in vivo, suggesting telomerase inhibition as an effective strategy for preventing MPN progression. METHODS To quantify hTERT level and ADAR1 activity in the setting of normal HSPC and MPN stem cell evolution, whole genome sequencing (WGS) analysis was performed on 76 normal and MPN blood CD34+ cells and matching saliva samples. Results were compared with RNA-seq of 100 FACS purified young, aged, MPN and AML CD34+CD38- stem cells and CD34+CD38+ progenitor cells. Confocal fluorescence microscopic evaluation of stem cell ADAR1 and hTERT localization, telomere length by Flow-FISH and telomerase activity by TRAP assays, lentiviral ADAR1 overexpression and shRNA knockdown were performed. In vitro stromal co-cultures, and humanized immunocompromised mouse models were established to determine the impact of imetelstat (a oligonucleotide inhibitor of telomerase) on normal, MPN stem cell and LSC maintenance. RESULTS Combined hTERT overexpression, ADAR1 activation and a significant reduction in telomere length correlated with accelerated stem cell aging during MPN progression to AML. Increased ADAR1 mediated adenosine to inosine (A-to-I) transcript editing coincided with accelerated telomere shortening in high risk MPN stem cells. Moreover, lentiviral ADAR1 overexpression enhanced pre-LSC engraftment. Treatment with imetelstat reduced MPN stem cell and LSC propagation in stromal co-cultures as well as in humanized mouse models commensurate with reduced hTERT expression levels and telomerase activity and decreased ADAR1 editing activity. Specifically, stromal co-culture assays revealed that combined treatment with dasatinib at 1 nM, and imetelstat at 1 µM or 5 µM significantly inhibited survival and replating of blast crisis (BC) CML progenitors compared with aged bone marrow progenitors (p &lt; 0.001, ANOVA). As a single agent, imetelstat (5 µM) inhibited survival and replating of pre-LSC derived from myelofibrosis compared with normal bone marrow progenitor samples (p &lt; 0.001, ANOVA). In pre-LSC MPN NSG-SGM mouse models established from 4 different MF samples, a significant reduction in proliferation of human CD45+ cells (p &lt; 0.01, t test) was observed in bone marrow and spleen, when compared with vehicle control. Treatment of humanized LSC mouse models, established with 5 different BC CML, with 30 mg/kg of imetelstat, 3 times a week for 4 weeks resulted in a significant reduction in proliferation of malignant progenitors and human CD45+ cells (p &lt; 0.001, ANOVA). As measured by a Flow-FISH assay, abnormal telomere length was reversed by imetelstat treatment compared with mismatch control (p &lt; 0.05, ANOVA). In addition, FACS analysis revealed a significant reduction in activated beta-catenin expression after imetelstat treatment of LSC engrafted mice compared with vehicle control (p &lt; 0.01, ANOVA). Finally, RNA-seq analysis performed on human CD34+ cells from imetelstat treated LSC mouse models revealed a significant reduction in LSC harboring malignant ADAR1-mediated A-to-I editing at doses that spared normal hematopoietic stem cells. CONCLUSIONS Combined WGS and RNA-Seq analyses, lentiviral ADAR1 overexpression, stromal co-culture assays and humanized pre-LSC and LSC mouse model studies reveal that pre-LSC evolution into LSC coincides with both ADAR1 and hTERT activation, which can be prevented with imetelstat. Disclosures Rizo: Geron Corp: Current Employment, Current equity holder in publicly-traded company. Huang:Geron Corp: Current Employment, Current equity holder in publicly-traded company. Jamieson:Forty Seven Inc: Patents & Royalties; Bristol-Myers Squibb: Other.

Telomerase activity, mTert gene expression and the telomere length in mouse mesenchymal stem cells in the late period after γ- and γ,n-irradiation and in the tumors developed from these cells

2020 ◽  
Vol 66 (3) ◽  
pp. 265-273
Author(s):  
O.V. Vysotskaya ◽  
A.I. Glukhov ◽  
Yu.P. Semochkina ◽  
S.A. Gordeev ◽  
E.Yu. Moskaleva
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Telomere Length ◽  
Cell Lines ◽  
Telomerase Activity ◽  
Mouse Bone Marrow ◽  
Fibrosarcoma Cell

In proliferating normal and tumor cells, the telomere length (TL) is maintained by high telomerase activity (TA). In the absence of TA the TL maintenance involves a mechanism of alternative lengthening of telomeres (ALT). The aim of this study was to investigate the level of TA, the mTert expression and TL in cultured normal and transformed by γ- and γ,n-irradiation mesenchymal stem cells (MSCs) from mouse bone marrow, in sarcomas that developed after the transplantation of these cells into syngeneic mice, and in fibrosarcoma cell lines obtained from these tumors to find out the role of AT or ALT in maintaining TL in these cells. During prolonged cultivation of normal and transformed under the influence of γ- (1 Gy and 6 Gy) and γ,n-irradiation (0.05 Gy, 0.5 Gy, and 2 Gy) MSCs from mouse bone marrow, a decrease in TA was detected in irradiated cells. Even deeper decrease in TA was found in sarcomas developed after administration of transformed MSCs to syngeneic mice and in fibrosarcoma cell lines isolated from these tumors in which TA was either absent or was found to be at a very low level. TL in three of the four lines obtained was halved compared to the initial MSCs. With absent or low TA and reduced TL, the cells of all the obtained fibrosarcoma lines successfully proliferated without signs of a change in survival. The mechanism of telomere maintainance in fibrosarcoma cell lines in the absence of TA needs further investigation and it can be assumed that it is associated with the use of the ALT. The detected decrease or absence of TA in transformed under the action of irradiation MSCs with the preservation or even an increase in the telomerase gene expression may be associated with the formation of inactive splicing variants, and requires further study. The obtained lines of transformed MSCs and fibrosarcomas with TA and without the activity of this enzyme can be a useful model for studying the efficacy of TA and ALT inhibitors in vitro and in vivo.

Telomere length and telomerase activity during expansion and differentiation of human mesenchymal stem cells and chondrocytes

2004 ◽  
Vol 82 (1) ◽  
pp. 49-55 ◽  
Author(s):  
J�rg Fellenberg ◽  
Tim H. Br�mmendorf ◽  
Anna-Maria Eschlbeck ◽  
Wiltrud Richter ◽  
Dominik Parsch
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Telomere Length ◽  
Human Mesenchymal Stem Cells ◽  
Telomerase Activity

scholarly journals Cannabidiol and Vitamin D3 Impact on Osteogenic Differentiation of Human Dental Mesenchymal Stem Cells

Medicina ◽  
2020 ◽  
Vol 56 (11) ◽  
pp. 607
Author(s):  
Nausica B. Petrescu ◽  
Ancuta Jurj ◽  
Olga Sorițău ◽  
Ondine P. Lucaciu ◽  
Noemi Dirzu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Osteogenic Differentiation ◽  
Vitamin D3 ◽  
Bone Matrix ◽  
Third Molar ◽  
Osteogenic Potential ◽  
Rt Pcr ◽  
Alizarin Red

Background and objective: The aim of the present study was to establish a new differentiation protocol using cannabidiol (CBD) and vitamin D3 (Vit. D3) for a better and faster osteogenic differentiation of dental tissue derived mesenchymal stem cells (MSCs). Materials and methods: MSCs were harvested from dental follicle (DFSCs), dental pulp (DPSCs), and apical papilla (APSCs) of an impacted third molar of a 17-year old patient. The stem cells were isolated and characterized using flow cytometry; reverse transcription polymerase chain reaction (RT-PCR); and osteogenic, chondrogenic, and adipogenic differentiation. The effects of CBD and Vit. D3 on osteogenic differentiation of dental-derived stem cell were evaluated in terms of viability/metabolic activity by alamar test, expression of collagen1A, osteopontin (OP), osteocalcin (OC), and osteonectin genes and by quantification of calcium deposits by alizarin red assay. Results: Stem cell characterization revealed more typical stemness characteristics for DFSCs and DPSCs and atypical morphology and markers expression for APSCs, a phenotype that was confirmed by differences in multipotential ability. The RT-PCR quantification of bone matrix proteins expression revealed a different behavior for each cell type, APSCs having the best response for CBD. DPSCs showed the best osteogenic potential when treated with Vit. D3. Cultivation of DFSC in standard stem cell conditions induced the highest expression of osteogenic genes, suggesting the spontaneous differentiation capacity of these cells. Regarding mineralization, alizarin red assay indicated that DFSCs and APSCs were the most responsive to low doses of CBD and Vit. D3. DPSCs had the lowest mineralization levels, with a slightly better response to Vit. D3. Conclusions: This study provides evidence that DFSCs, DPSCs, and APSCs respond differently to osteoinduction stimuli and that CBD and Vit. D3 can enhance osteogenic differentiation of these types of cells under certain conditions and doses.

scholarly journals Comparison of senescence-related changes between three- and two-dimensional cultured adipose-derived mesenchymal stem cells

2020 ◽  
Author(s):  
Qiliang Yin ◽  
Na Xu ◽  
Dong sheng Xu ◽  
Ming xin Dong ◽  
Xiu min Shi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Energy Metabolism ◽  
Cell Growth ◽  
Telomere Length ◽  
Cell Morphology ◽  
Telomerase Activity ◽  
Two Dimensional ◽  
3D Cultures ◽  
2D And 3D

Abstract Background: Adipose-derived mesenchymal stem cells ( ADMSCs ) have attracted widespread interest as cell-based tissue repair systems. To obtain adequate quantities of ADMSCs for therapeutic applications, extensive in vitro expansion is required. However, under current two-dimensional (2D) approaches , ADMSCs rapidly undergo replicative senescence , and cell growth is impeded and stem cell properties are eliminated by mechanisms that are poorly understood . These issues limit the extensive applications of ADMSCs . In this study, we investigated senescence-related changes in mesenchymal stem cells (MSCs) isolated from human adipose tissue in 2D and three-dimensional (3D) cultures. Methods: We studied cell growth over a given period (21 days) to determine if modes of culture were associated with ADMSCs senescence . ADMSCs were isolated from healthy females by liposuction surgery and then were grew in 2D and 3D cultures. The cell morphology was observed during cell culture. Every other time of culture, senescence-associated β-galactosidase (SA-β-gal) expression, cell viability, proliferation, and differentiation potential of ADMSCs from 2D and 3D cultures were detected. Also, senescence and stemness related genes expression, telomere length, telomerase activity, and energy metabolism of ADMSCs for different culture time were evaluated. Results: With long-term propagation, we observed significant changes in cell morphology, proliferation, differentiation abilities and energy metabolism, which were associated with increases in SA-β-gal activity, and decreases in telomere length and telomerase activity . Notably, when cultured in 3D, these changes were improved. Conclusions: Our results indicate that 3D culture is able to ameliorate senescence-related changes in ADMSCs.

scholarly journals Interleukin-6, -8, and TGF-β Secreted from Mesenchymal Stem Cells Show Functional Role in Reduction of Telomerase Activity of Leukemia Cell Via Wnt5a/β-Catenin and P53 Pathways

2020 ◽  
Vol 10 (2) ◽  
pp. 307-314 ◽  
Author(s):  
Ezzatollah Fathi ◽  
Behnaz Valipour ◽  
Zohreh Sanaat ◽  
Hojjatollah Nozad Charoudeh ◽  
Raheleh Farahzadi
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Telomere Length ◽  
Real Time Pcr ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Telomerase Activity ◽  
Htert Gene ◽  
Gene And Protein Expression

Purpose: The effect of mesenchymal stem cells (MSCs) on the immortality features of malignant cells, such as hematologic cancerous cells, are controversial, and the associated mechanisms are yet to be well understood. The aim of the present study was to investigate the in vitro effect of bone marrow-derived MSCs (BMSCs) on the chronic myeloid leukemia cell line K562 through telomere length measurements, telomerase activity assessments, and hTERT gene expression. The possible signaling pathways involved in this process, including Wnt-5a/β-catenin and P53, were also evaluated. Methods: Two cell populations (BMSCs and K562 cell line) were co-cultured on transwell plates for 7 days. Next, K562 cells were collected and subjected to quantitative real-time PCR, PCR-ELISA TRAP assay, and the ELISA sandwich technique for telomere length, hTERT gene expression, telomerase activity assay, and cytokine measurement, respectively. Also, the involvement of the mentioned signaling pathways in this process was reported by real-time PCR and Western blotting through gene and protein expression, respectively. Results: The results showed that BMSCs caused significant decreases in telomere length, telomerase activity, and the mRNA level of hTERT as a regulator of telomerase activity. The significant presence of interleukin (IL)-6, IL-8, and transforming growth factor beta (TGF-β) was obvious in the co-cultured media. Also, BMSCs significantly decreased and increased the gene and protein expression of β-catenin and P53, respectively. Conclusion: It was concluded that the mentioned effects of IL-6, IL-8, and TGF-β cytokines secreted from MSCs on K562 cells as therapeutic agents were applied by Wnt-5a/β-catenin and P53 pathways

scholarly journals ID: 1051 Telomerase activity, telomere length and P53 mutation detection on cellular senescence of Human Amnion Mesenchymal Stem Cells (HAMCs)

2017 ◽  
Vol 4 (S) ◽  
pp. 131
Author(s):  
Fiona Macniesia Thomas ◽  
Vijay Kumar ◽  
Siti Fatimah Simat ◽  
Helen Benedict Lasimbang
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Telomere Length ◽  
Cellular Senescence ◽  
Mutation Detection ◽  
Telomerase Activity ◽  
P53 Mutation ◽  
Human Amnion ◽  
Long Term Culture

A fundamental understanding of senescence in human amnion mesenchymal stem cells (HAMCs) is crucial for its application in cellular therapy. Previous findings strongly support that HAMCs undergoes cellular senescence after long term in-vitro culture, with evidence of significant morphological changes and the presence of the senescent associated β-galactosidase (SA-β-Gal) marker. The telomere length and the telomerase activity have been linked with cellular aging and they are important in regulating cell proliferation. In addition, p53 gene has been associated with cell senescence. The aim of this study was to investigate the telomerase activity, telomere length in senescent HAMCs, and to detect p53 mutations in these cells. Samples were obtained from amnion placenta and then cultured for long term. Prolong-cultured HAMCs was isolated at passages 5, 10 and 15 and then analysed via telomeric repeat amplification protocol (TRAP), telomere length assay and p53 mutation detection assay. The results showed that after long term culture of HAMCs, there was a decrease in telomere length and telomerase activity from passages 5, 10 to 15. Telomerase controls the telomere’s length which maintains the cells proliferation. The decrease of telomere length and telomerase activity may suggest that the proliferation of HAMCs has slowed down due to HAMCs entering senescence after long term culture. P53 mutation detection study indicated that HAMCs at all passage did not have altered sequences. Thus, the cells did not undergo uncontrollable replication due to the effect of long-term culture. Further studies on senescence in HAMCs will be assessed by investigating the expression level of p53, p21, p16, pRB and GADD45 genes in long term culture of HAMCs via RT-qPCR. The findings will help us understand the associations between gene expressions and the process of senescence

scholarly journals Molecular Signatures of Tumor-Initiating Cells Unveil Wnt Pathway As a Therapeutic Target in Mantle Cell Lymphoma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2148-2148 ◽  
Author(s):  
Felipe Samaniego ◽  
Lalit Sehgal ◽  
Frank K Braun ◽  
Zuzana Berkova ◽  
Jorge E. Romaguera ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Wnt Signaling ◽  
Mantle Cell Lymphoma ◽  
Wnt Pathway ◽  
Cell Lymphoma ◽  
Rt Pcr ◽  
Mantle Cell

Abstract Introduction Mantle cell lymphoma (MCL) is an aggressive and incurable form of non-Hodgkin’s lymphoma. Despite initial responses to intense chemotherapy, up to 50% of cases of MCL relapse, often in a chemoresistant form. We hypothesized that the recently identified MCL-initiating cells (MCL-ICs) are the main reason for relapse and chemoresistance of MCL. Methods We isolated MCL-ICs from primary MCL cells on the basis of a defined marker expression pattern; CD34-CD3-CD45+CD19-. The MCL-ICs, MCL-non-ICs, and peripheral blood lymphocytes from healthy donors were analyzed for gene expression using the Arraystar platform. The differences in mRNA levels of genes of interest were confirmed by quantitative RT-PCR. The prominent differentially expressed transcripts were analyzed using the Ingenuity Platform. Primary MCL cells were co-culture with mesenchymal stem cells to assess the effects of chemotherapeutic agents such as vincristine, doxorubicin and the newly approved Burton tyrosine kinase inhibitor ibrutinib, and Wnt signaling inhibitors. Results Approximately 1% of primary MCL cells are MCL-ICs and they can be maintained in co-culture with mesenchymal stem cells. Comparison of gene expression profiles of MCL-ICs and MCL-non-ICs revealed activation of stem cell-specific pathways in MCL-ICs by expression of Wnt, Notch, and Hedgehog and enhanced expression of Nanog, Oct4, KLF4, ADH1, MT1b and ABCC3. Gene expression microarray data and RT-PCR data suggested predominant activation of the Wnt/Frizzled pathway. Indeed, MCL-ICs were particularly sensitive to Wnt pathway inhibitors. Targeting Wnt-dependent β-catenin‒TCF4 interaction with CCT036477, iCRT14, or PKF118-310 preferentially eliminated the MCL-ICs, reduced the expression of stem cell transcription factors (Myc, Nanog, Oct4, Klf4), and sensitized MCL cells to vincristine, doxorubicin, and ibrutinib. Interestingly, while vincristine, doxorubicin or ibrutinib did kill MCL cells, they did not reduce the percentage of MCL-ICs in treated co-culture. Conclusion MCL-ICs are present in primary MCL isolates and they show gene expression pattern of chemoresistant, stem cell-like cells with predominant activation of Wnt signaling. In order to produce durable remissions in MCL patients, treatment strategies should be directed to target MCL-ICs. Disclosures Wang: Pharmacyclics, Janssen: Honoraria, Research Funding.

scholarly journals Experimental Study of lncRNA RP11-815M8.1 Promoting Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells

2021 ◽  
Vol 2021 ◽  
pp. 1-8
Author(s):  
Xiang Sun ◽  
Junchuan Cao ◽  
Jiusong Han ◽  
Bo Jia ◽  
Jing Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Transcription Factors ◽  
Osteogenic Differentiation ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Rt Pcr ◽  
Alizarin Red ◽  
Marrow Mesenchymal Stem Cells

Objective. This study is aimed at investigating the role of long noncoding RNA (lncRNA) RP11-815M8.1 in the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). Methods. RT-PCR was used to detect the expression of lncRNA RP11-815M8.1 before and after osteogenic differentiation of hBMSCs. The lncRNA RP11-815M8.1 in hBMSCs was overexpressed or silenced via lentiviral transfection. The transfection efficiency was detected by RT-PCR, and the proliferation of hBMSCs was determined by CCK-8. After 14 days of osteogenic differentiation of transfected hBMSCs, the expression of osteogenic transcription factors (ALP, OCN, OPN, Runx2, and Osterix) was detected by alizarin red staining and RT-PCR. The mRNAs directly regulated by lncRNA RP11-815M8.1 and targeted miRNAs were analyzed according to the positional relationship between lncRNA and mRNA in the genome and miRanda software. Results. The expression of lncRNA RP11-815M8.1 enhanced with increasing osteogenic differentiation time of hBMSCs. Two days after the transfection of hBMSCs, lncRNA RP11-815M8.1 expression was significantly increased in the overexpression group and significantly decreased in the knockdown group, compared to control cells. The CCK-8 assay showed that overexpression and knockdown of lncRNA RP11-815M8.1 did not affect the proliferation of hBMSCs. After 14 days of differentiation of hBMSCs, stronger alizarin red staining was observed in the overexpression groups, and the expression of osteogenic transcription factors was increased in the overexpression group compared to the control. In the knockdown group, alizarin red staining and the expression of osteogenic transcription factors were decreased. Bioinformatics analysis showed that lncRNA RP11-815M8.1 was directly associated with one mRNA, 27 interacting miRNAs, and 20 miRNA-targeted mRNAs. Conclusion. The osteogenic differentiation of hBMSCs can be promoted by lncRNA RP11-815M8.1 in vitro.

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