OBJECTIVE: To investigate the changes and functions of Sox2 gene expression and promoter methylation during induced differentiation of bone marrow mesenchymal stem cells (BMSCs) into hepatocytes (HCs).
METHODS: Rat bone marrow Thy-1+Lin- cells were prepared and divided into control group (directed induction of differentiation into HCs) and experimental group (5-azacytidine intervention induced differentiation). The mRNA expression levels of ALB and Sox2 were detected by fluorescence quantitative polymerase chain reaction (PCR), and the Sox2 gene promoter methylation level was determined by Bisulfite sequencing PCR (BSP).
RESULTS: Sox mRNA expression level was significantly increased in experimental group compared to the control group at 0, 7, and 14 days, respectively (all P<0.05). The Sox2 promoter methylation level was gradually increased after 0, 7 and 14 days induction in both groups, accompanied by an increase in methylated loci (all P<0.05). Statistical significance was present in CpG methylated loci between groups (all P<0.05).
CONCLUSIONS: The expression of Sox2 gene increased first and then decreased in the process of inducing rat BMSCs into stem cells, and the methylation level of CpG loci in the promoter region changed dynamically, with an increased overall methylation level. After 5-aza treatment, the Sox2 promoter was in a non-methylated state, and its mRNA expression increased, which hindered the cell differentiation.

To adapt to the extreme conditions of plateau environments, yaks have evolved thick hair, making them an ideal model for investigating the mechanisms involved in hair growth. We can gain valuable insights into how hair follicles develop and their cyclic growth in challenging environments by studying yaks. However, the lack of essential data on yak hair follicle histology and the absence of in vitro cell models for hair follicles serve as a limitation to such research objectives. In this study, we investigated the structure of skin tissue during different hair follicle cycles using the yak model. Additionally, we successfully established in vitro models of hair follicle-associated cells derived from yak skin, including dermal papilla cells (DPCs), preadipocytes, and fibroblasts. We optimized the microdissection technique for DPCs culture by simplifying the procedure and reducing the time required. Furthermore, we improved the methodology used to differentiate yak preadipocytes into mature adipocytes, thus increasing the differentiation efficiency. The introduction of yak as a natural model provides valuable research resources for exploring the mechanisms of hair growth and contributes to a deeper understanding of hair follicle biology and the development of regenerative medicine strategies.

In a preliminary experiment, it was found that c-myc expression was decreased following the differentiation of THP-1 cells into monocytes/macrophages induced by phorbol 12-myristate 13 acetate (PMA) + lipopolysaccharide (LPS) + interferon (IFN)-γ. The expression of miR-let-7c-5p was then found to be elevated by cross-sectional analysis using TargetScan and PubMed and differential microarray analysis. The present study aimed to investigate the role of the miR-let-7c-5p/c-myc signaling axis in the committed differentiation of THP-1 leukemic cells into monocytes/macrophages induced by PMA + LPS + IFN-γ. Human THP-1 leukemic cells were induced to differentiate into monocytes/macrophages by PMA + LPS + IFN-γ. Following induction for 48 h, the growth density of the THP-1 cells was observed directly under an inverted microscope, cell proliferation was measured using Cell Counting Kit-8 assay and the cell cycle and the expression of differentiation-related antigens (CD11b and CD14) were measured using flow cytometry. The mRNA expression of miR-let-7c-5p and c-myc was detected using reverse transcription-quantitative PCR and the protein expression of c-myc was detected using western blot analysis. Dual luciferase reporter gene analysis was used to detect the targeted binding of miR-let-7c-5p on the 3\'UTR of c-myc. The relative expression of miR-let-7c-5p and c-myc genes in THP-1 cells induced by PMA + LPS + IFN-γ was found to be up- and downregulated respectively, and expression of miR-let-7c-5p was negatively correlated with the expression of c-myc gene. Dual luciferase reporter gene assays confirmed that miR-let-7c-5p targeted the 3\'UTR of c-myc and inhibited luciferase activity. Following transfection with miR-let-7c-5p mimics, the expression of c-myc was markedly downregulated and the proliferative ability of the THP-1 cells was decreased, while the expression rate of CD11b and CD14 was significantly increased. The rescue experiment revealed that the effects of miR-let-7c-5p mimics on the proliferation and differentiation of THP-1 cells were attenuated by transfection with c-myc overexpression vector. Together, the findings of the present study demonstrated that miR-let-7c-5p can target the 3\'UTR region of c-myc and that the miR-let-7c-5p/c-myc signaling axis is one of the critical pathways involved in the directional differentiation of leukemic cells into monocytes/macrophages.

Hematopoietic stem and progenitor cell (HSPC) research will help elucidate the pathogenesis of hematologic diseases. The present study aimed to establish an isolation method and culture system for chicken bone marrow (BM)-derived HSPCs and test their proliferation and differentiation abilities. Mononuclear cells were collected from chicken BM, and CD34+ HSPCs were isolated. Then, the cells were cultured in media with different cytokine compositions, and the growth status, cell phenotype, and morphological appearance of the cells were analyzed at different time points. Our results showed that Iscove\'s Modified Dulbecco\'s Medium supplemented with 50 ng/mL stem cell factor, 30 ng/mL Flt-3 ligand, 10 μg/mL interleukin 3, 50 ng/mL interleukin 6%, and 10% chicken serum supported chicken CD34+ HSPC survival ex vivo for approximately 10 d. Further, 80 ng/mL granulocyte-colony stimulating factor and 30 ng/mL granulocyte macrophage-colony stimulating factor were added into the above culture system to form a myeloid cell differentiation induction culture system. After culturing in this system for 72 h, approximately 66% of chicken CD34+ HSPCs exhibited a CD11b+ phenotype, indicating that HSPCs differentiated into myeloid cells. In conclusion, chicken BM-derived CD34+ cells possess HSPC characteristics that can self-renew and differentiate into myeloid cells in a culture medium containing growth factors.

The purpose of this study was to clone and characterize the ZFP36L1 (zinc finger protein 36-like 1) gene, clarify its expression characteristics, and elucidate its expression patterns in different tissues of goats. Samples of 15 tissues from Jianzhou big-eared goats, including heart, liver, spleen, lung and kidney were collected. Goat ZFP36L1 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR), then the gene and protein sequence were analyzed by online tools. Quantitative real-time polymerase chain reaction (qPCR) was used to detect the expression level of ZFP36L1 in intramuscular preadipocytes in different tissues and adipocytes of goat at different differentiation stages. The results showed that the length of ZFR36L1 gene was 1 224 bp, and the coding sequence (CDS) region was 1 017 bp, encoding 338 amino acids, which was a non-secretory unstable protein mainly located in nucleus and cytoplasm. Tissue expression profile showed that ZFP36L1 gene was expressed in all selected tissues. In visceral tissues, the small intestine showed the highest expression level (P < 0.01). In muscle tissue, the highest expression level was presented in longissimus dorsi muscle (P < 0.01), whereas the expression level in subcutaneous adipose tissue was significantly higher than that in other tissues (P < 0.01). The results of induced differentiation showed that the expression of this gene was up-regulated during adipogenic differentiation of intramuscular precursor adipocytes (P < 0.01). These data may help to clarify the biological function of the ZFP36L1 gene in goat.

OBJECTIVE: To detect mRNA and protein expression of meiosis-specific genes in human umbilical cord mesenchymal stem cells (hUMSCs) in an in vitro co-culture microenvironment with mouse primordial germ cells (PGCs), and to further explore the effective potential of hUMSCs to differentiate into PGCs.
METHODS: HUMSCs were obtained from human Wharton\'s jelly fragments by adherent culture. PGCs were derived from 12.5 days post-coitum (dpc) BalbC mice. Then hUMSCs were co-cultured with PGCs in Matrigel, inside or outside of a culture chamber, respectively. The changes in morphology and cytogenetic characteristics were observed. SCP3 and DDX4 expression in hUMSCs were detected and analyzed using immunofluorescence staining. Oct-4, Stra8, Zp3 and Dmc1 gene expressions in PGCs, hUMSCs, and hUMSCs after co-culture with PGCs were analyzed by real time reverse transcription-polymerase chain reaction.
RESULTS: Both hUMSCs and PGCs expressed Oct-4 at different degrees. After co-culture with PGCs, hUMSCs became rounded and showed AKP activity. HUMSCs suspension-cultured in Matrigel or adherent cultured with cell chamber significantly expressed Stra8, DMC1, SCP3 and DDX4 genes.
CONCLUSIONS: HUMSCs can be induced to express PGC-specific genes Stra8 and DMC1, spermatogonium/oogonium-specific genes SCP3 and DDX4 that predict directed differentiation potential into early germ cells at a molecular level.

Studies of induced granulocytic differentiation help to reveal molecular mechanisms of cell maturation. The nuclear proteome represents a rich source of regulatory molecules, including transcription factors (TFs). It is important to have an understanding of molecular perturbations at the early stages of the differentiation processes. By applying the proteomic quantitative profiling using isobaric labeling, we found that the contents of 214, 319, 376, 426, and 391 proteins were altered at 3, 6, 9, 12, and 72 h, respectively, compared to 0 h in the HL-60 cell nuclear fraction under all-trans-retinoid acid (ATRA) treatment. From 1860 identified nuclear proteins, 231 proteins were annotated as proteins with transcription factor (TF) activity. Six TFs (RREB1, SRCAP, CCDC124, TRIM24, BRD7, and BUD31) were downregulated and three TFs EWSR1, ENO1, and FUS were upregulated at early time points (3-12 h) after ATRA treatment. Bioinformatic annotation indicates involvement of the HL-60 nuclear proteome in DNA damage recognition in the RUNX1-triggered pathway, and in the p53-regulation pathway. By applying scheduled multiple reaction monitoring using stable isotopically labeled peptide standards (MRM/SIS), we found a persistent increase in the content of the following proteins: PRAM1, CEPBP, RBPJ, and HIC1 in the HL-60 cell nuclear fraction during ATRA-induced granulocytic differentiation. In the case of STAT1, CASP3, PARP1, and PRKDC proteins, a transient increase in their content was observed at early time points (3-12 h) after the ATRA treatment. Obtained data on nuclear proteome composition and dynamics during granulocytic differentiation could be beneficial for the development of new treatment approaches for leukemias with the mutated p53 gene.

The aim of this study was to investigate the effect of activating transcription factor 3 (ATF3) on the differentiation of intramuscular preadipocytes in goat, and to elucidate its possible action pathway at the molecular level. In this study, the recombinant plasmid of goat pEGFP-N1-ATF3 was constructed, and the intramuscular preadipocytes were transfected with liposomes. The relative expression levels of adipocyte differentiation marker genes were detected by quantitative real-time PCR (qRT-PCR). After transfection of goat intramuscular preadipocytes with the goat pEGFP-N1-ATF3 overexpression vector, it was found that the accumulation of lipid droplets was inhibited, and the adipocyte differentiation markers PPARγ, C/EBPα and SREBP1 were extremely significantly down-regulated (P < 0.01), while C/EBPβ and AP2 were significantly down-regulated (P < 0.05). The ATF3 binding sites were predicted to exist in the promoter regions of PPARγ, C/EBPα and AP2 by the ALGGEN PROMO program. The overexpression of goat ATF3 inhibits the accumulation of lipid droplets in intramuscular preadipocytes, and this effect may be achieved by down-regulating PPARγ, C/EBPα and AP2. These results may facilitate elucidation of the regulatory mechanism of ATF3 in regulating the differentiation of goat intramuscular preadipocytes.

Multiple methodologies have been reported to facilitate skin-derived precursor (SKP) growth, but the impact of plating density on SKP growth has not been studied. To determine the optimal plating density, we used six plating densities and two types of flasks for mouse SKP (mSKP) culture. On the 14th day, the number, diameter, and viability of mSKP spheres were compared by morphological assessment and cell counting kit 8, and we found the optimal plating density was 2.5 × 105-5 × 105 cells/mL. In addition, we investigated the correlation between the SKP spheres and the adherent cell colonies in the serum-free culture system. We treated the adherent cell colonies with two culture conditions and characterized the cells generated from two conditions by immunocytochemistry and induced differentiation, respectively. The results elucidated that the adherent cell colonies differentiated into either mSKPs or dermal mesenchymal stem cells under appropriate culture conditions. In conclusion, mSKP spheres differentiated from the adherent cell colonies. The optimal plating density significantly promoted and advanced the proliferation of adherent cell colonies, which optimized mSKP growth and yield. The adherent cell colonies possessed the capacity of differentiating into different types of cells under appropriate culture conditions.

Induced granulocytic differentiation of human leukemic cells under all-trans-retinoid acid (ATRA) treatment underlies differentiation therapy of acute myeloid leukemia. Knowing the regulation of this process it is possible to identify potential targets for antileukemic drugs and develop novel approaches to differentiation therapy. In this study, we have performed transcriptomic and proteomic profiling to reveal up- and down-regulated transcripts and proteins during time-course experiments. Using data on differentially expressed transcripts and proteins we have applied upstream regulator search and obtained transcriptome- and proteome-based regulatory networks of induced granulocytic differentiation that cover both up-regulated (HIC1, NFKBIA, and CASP9) and down-regulated (PARP1, VDR, and RXRA) elements. To verify the designed network we measured HIC1 and PARP1 protein abundance during granulocytic differentiation by selected reaction monitoring (SRM) using stable isotopically labeled peptide standards. We also revealed that transcription factor CEBPB and LYN kinase were involved in differentiation onset, and evaluated their protein levels by SRM technique. Obtained results indicate that the omics data reflect involvement of the DNA repair system and the MAPK kinase cascade as well as show the balance between the processes of the cell survival and apoptosis in a p53-independent manner. The differentially expressed transcripts and proteins, predicted transcriptional factors, and key molecules such as HIC1, CEBPB, LYN, and PARP1 may be considered as potential targets for differentiation therapy of acute myeloid leukemia.