Autologous or artificial bone grafts have been widely used to repair maxillofacial bone defects clinically, but these methods still suffer from insufficient osteogenesis. Bone marrow mesenchymal stem cells play a key role in the bone formation. Notably, ectoderm-derived jaw bone marrow mesenchymal stem cells have stronger proliferation and osteogenic differentiation capacity compared with mesoderm-derived iliac bone marrow mesenchymal stem cells, elucidating the key mechanisms involved. It is expected to provide a new strategy for the repair of craniomaxillofacial bone defects.

To compare the biological differences between human jaw bone marrow mesenchymal stem cells and iliac bone marrow mesenchymal stem cells and identify the key regulatory genes.

(1) Jaw bone and iliac bone were collected from three patients with alveolar cleft. Primary bone marrow mesenchymal stem cells were isolated and cultured. Cell proliferation ability was detected by colony formation assay. Cell senescence was detected by β-galactosidase staining assay. Senescence and osteogenesis-related protein expression levels were detected by western blot assay. Osteogenic ability was detected by alizarin red staining after osteogenic induction solution treatment. (2) Jaw bone marrow mesenchymal stem cells and iliac bone marrow mesenchymal stem cells were subjected to transcriptome and differential gene expression analysis to find the 20 genes with the largest differential expression and identify the key regulatory factors. (3) The gene in iliac bone marrow mesenchymal stem cells were knocked down to comparatively analyze the changes in self-renewal, anti-aging and osteogenic capacity of iliac bone marrow mesenchymal stem cells. (4) The gene-edited iliac bone marrow mesenchymal stem cells were loaded into β-tricalcium phosphate scaffolds and implant into nude mice for 8 weeks. The scaffolds were stained with Masson staining and immunofluorescence staining to observe the difference in osteogenic capacity.

(1) Jaw bone marrow mesenchymal stem cells have stronger proliferation, anti-aging and osteogenic differentiation abilities compared to iliac bone marrow mesenchymal stem cells. (2) By transcriptome analysis, we identified HOXA10 as a highly up-regulated core transcription factor in iliac bone marrow mesenchymal stem cells. (3) After knocking down HOXA10 in iliac bone marrow mesenchymal stem cells, we observed a significant increase in proliferation, anti-aging, and osteogenic differentiation abilities. (4) After HOXA10 knocked-down iliac bone marrow mesenchymal stem cells/β-tricalcium phosphate was implanted subcutaneously on the back of nude mice, and their bone formation ability was stronger. (5) The above results suggest that HOXA10 is a key regulatory gene that determines the proliferative, anti-aging and osteogenic differentiation abilities of bone marrow mesenchymal stem cells. HOXA10 gene-modified iliac bone marrow mesenchymal stem cell transplantation can be used as a potential application strategy for repairing maxillofacial bone defects.

Bone marrow mesenchymal stem cells are the main effector cells for bone formation. With the increase of age, the regenerative ability of bone marrow mesenchymal stem cells is weakened and the differentiation function is impaired, leading to poor osteoporosis. Therefore, restoring the regenerative capacity and cellular function of aged bone marrow mesenchymal stem cells is essential for the effective treatment of osteoporosis.

To investigate the effects of passage 3 and passage 11 bone marrow mesenchymal stem cells-derived exosomes of young rats on the aging of bone marrow mesenchymal stem cells derived from elderly rats.

Bone marrow mesenchymal stem cells from 6-8-week-old female SD rats were isolated and cultured, and passaged to the passages 3 and 11, respectively. Then, exosomes from passages 3 and 11 bone marrow mesenchymal stem cells were extracted. Bone marrow mesenchymal stem cells from 18-month-old female SD rats were isolated and cultured, passaged to passage 3, and divided into 3 groups. The control group was routinely cultured, and the other two groups were intervened with exosomes from passages 3 and 11 bone marrow mesenchymal stem cells. After 48 hours of exosome intervention, the expression of β-galactosidase in the nucleus was detected by β-galactosidase staining kit. The expression of aging-related genes was detected by qRT-PCR. The expression differences of miRNA in exosomes from passages 3 and 11 bone marrow mesenchymal stem cells were compared by Small RNA sequencing.

(1) Compared with the control group and passage 11 bone marrow mesenchymal stem cell-derived exosomes group, the β-galactosidase activity of bone marrow mesenchymal stem cells of aged rats was significantly lower in the passage 3 bone marrow mesenchymal stem cell-derived exosomes group. (2) Compared with the control group, the expression of aging-related genes p21 and p16 was significantly reduced in the passage 3 bone marrow mesenchymal stem cell-derived exosome group (P < 0.05), while there was no significant difference in the expression of aging-related genes p21 and p16 in the passage 11 bone marrow mesenchymal stem cell-derived exosome group. (3) Sequencing results showed that there was a significant difference in the expression of miRNAs in the two exosomes, among which the miRNAs with the most significant expression differences were let-7c-5p, let-7b-5p, miR-320-3p, and miR-26a-5p. KEGG analysis results showed that significantly different miRNA enrichment pathways include mTOR, AMPK and other aging-related signaling pathways. The above results indicate that passage 3 bone marrow mesenchymal stem cell-derived exosomes have the ability to reverse the aging of bone marrow mesenchymal stem cells in aged rats.

With aging, the regenerative capacity and differentiation function of bone marrow mesenchymal stem cells progressively decline, reducing bone tissue repair efficacy. Thus, identifying bone marrow mesenchymal stem cell subpopulations with enhanced osteogenic potential is of significant importance for advancing bone tissue engineering.

To evaluate the osteogenic differentiation potential differences between STRO-1 positive and negative bone marrow mesenchymal stem cells under osteogenic induction conditions.

SD rat bone marrow mesenchymal stem cells were isolated and cultured. The expression of CD29, CD45, CD90, and STRO-1 was identified via flow cytometry and immunofluorescence. Immunomagnetic cell sorting was used to separate STRO-1 positive and negative bone marrow mesenchymal stem cells. The cells of two groups were subjected to osteogenic induction for 7 and 14 days. qRT-PCR and western blotting were performed to analyze differences in osteogenesis-related gene expression (Collagen I, Runt-related transcription factor 2, osteoprotegerin, and osteocalcin) and protein levels. Alizarin red staining and alkaline phosphatase staining were used to observe calcium nodule formation.

Flow cytometry showed high expression levels of CD29 and CD90 and low expression of CD45, with a positive STRO-1 expression rate of 12.8%. Immunofluorescence results were consistent with those of flow cytometry. After magnetic cell sorting, STRO-1 positive cells demonstrated a higher colony formation rate than STRO-1 negative cells. On day 14, STRO-1 positive cells showed significantly higher osteogenic differentiation potential than on day 7, with significantly elevated osteogenesis-related marker levels compared to STRO-1 negative cells (P < 0.01). The findings indicate that STRO-1 positive bone marrow mesenchymal stem cells exhibit significant advantages in osteogenic potential, providing a theoretical basis for their selection as ideal seed cells in bone tissue engineering. In future applications, they may represent a promising therapeutic approach for bone defect repair.

Osteoarthritis is a progressive joint condition identified by ongoing deterioration of the cartilage matrix, and there is currently no effective drug treatment plan. Metformin-modified exosomes isolated from bone marrow-derived mesenchymal stem cells can become a new method for treating osteoarthritis due to their avoidance of oral drug adverse reactions and immunogenicity.

To study the controlling impact of exosomes from metformin-altered bone marrow-derived mesenchymal stem cells on chondrocytes.

Rabbit bone marrow-derived mesenchymal stem cells and chondrocytes were cultured in vitro. Bone marrow-derived mesenchymal stem cells derived exosomes and metformin pretreated bone marrow-derived mesenchymal stem cells derived exosomes were collected using a high-speed centrifuge. Chondrocytes were cultured with exosome-containing culture medium for 24 hours and then treated with 100 µmol/L H₂O₂ for 24 hours. The capability changes of two extracellular vesicles on chondrocyte proliferation and migration were detected using CCK8 assay and scratch healing experiment, respectively. Western blot analysis and RT-qPCR were employed to examine the alterations in the expression of type II collagen, P16 protein, and their mRNA in chondrocytes.

Western blot analysis was utilized to assess the changes in the expression of MKK7/JNK pathway proteins. ELISA kits were utilized to measure the activity of cell superoxide dismutase and the levels of malondialdehyde in chondrocytes.

(1) In an oxidative stress environment, the proliferation and migration abilities of chondrocytes were weakened. The two types of exosomes could restore the proliferation and migration abilities of chondrocytes to a certain extent. Metformin pretreated bone marrow-derived mesenchymal stem cells derived exosomes had a significantly better improvement effect (P < 0.05). (2) Compared with normal bone marrow mesenchymal stem cell-derived exosomes, metformin pretreated bone marrow-derived mesenchymal stem cells derived exosomes could more effectively increase type II collagen expression and superoxide dismutase activity (P < 0.05), and were also more effective in reducing P16 expression and malondialdehyde levels (P < 0.05). (3) The two types of exosomes could inhibit the expression of MKK7 and p-JNK proteins to a certain extent, and the inhibitory effect of metformin pretreated bone marrow-derived mesenchymal stem cells derived exosomes was more significant (P < 0.05). The results show that in an oxidative stress environment, metformin pretreated bone marrow-derived mesenchymal stem cells derived exosomes resist chondrocyte aging and promote chondrocyte proliferation by inhibiting the MKK7/JNK pathway.

Previous studies have shown that Huosui Formula has a synergistic effect on the immune and hematopoietic regulation of patients with myelodysplastic syndrome, but the specific mechanism is not yet clear.

To explore the effect and mechanism of Huosui Formula on bone marrow hematopoiesis in rats with myelodysplastic syndrome.

A total of 70 SD rats were randomly divided into a normal control group (n=10), a model group (n=15), a western medicine group (n=15), a low-dose Huosui Formula group (n=15), and a high-dose Huosui Formula group (n=15). Except for the normal control group, the other four groups were injected with dimethylbenzanthracene via the tail vein to induce the establishment of rat myelodysplastic syndrome models. After modeling, the normal control group and the model group were given normal saline; the western medicine group was given thalidomide capsules 10 mg/kg and retinoic acid tablets 4 mg/kg, and the low-dose Huosui Formula group and the high-dose Huosui Formula group were given 1.5 and 6 g/kg Huosui Formula, respectively, by intragastric administration once a day for 28 consecutive days. Peripheral blood and femoral bone marrow tissue were collected to detect peripheral blood routine and bone marrow biopsy hematopoietic proliferation. Flow cytometry was used to detect T lymphocyte subsets and the expression of CTLA-4 and PD-1 on T lymphocytes.

(1) Compared with the normal control group, peripheral blood leukocyte, neutrophil, hemoglobin, platelet, and CD4⁺, CD4⁺/CD8⁺ levels were decreased in the model group significantly (P < 0.05), while CD4⁺PD-1⁺, CD8⁺PD-1⁺, CD4⁺CTLA-4⁺, and CD8⁺CTLA-4⁺ expressions were significantly upregulated (P < 0.05). (2) In all dosage groups, myelopoietic proliferation was increased compared with the model group, with no significant difference between the groups (P > 0.05). (3) Compared with the model group, leukocytes, hemoglobin, platelets, and CD4⁺, CD4⁺/CD8⁺ were significantly elevated in the high-dose Huosui Formula group (P < 0.05), the expression of CD8⁺ was significantly lower (P < 0.05), and the levels of CD4⁺PD-1⁺, CD8⁺PD-1⁺, CD4⁺CTLA-4⁺, and CD8⁺CTLA-4⁺ were down-regulated but not statistically significant (P > 0.05). (4) The western medicine group and the high-dose Huosui Formula group showed similar efficacy. The improvement of each index in the high-dose Huosui Formula group was superior to that in the low-dose Huosui Formula group. These findings indicate that Huosui Formula can improve the bone marrow hematopoiesis in myelodysplastic syndrome model rats, increase the levels of CD4⁺, and CD4⁺/CD8⁺ while down-regulate the expression levels of CD4⁺PD-1⁺, CD8⁺PD-1⁺, CD4⁺CTLA-4⁺, and CD8⁺CTLA-4⁺. These observations suggest a link to the negative immunoregulation mechanism.

Human umbilical cord mesenchymal stem cell-derived exosomes were found to be effective in promoting neural repair in spinal cord injury.

To investigate whether exosomes derived from human umbilical cord mesenchymal stem cells are able to attenuate neuroinflammation and promote recovery of motor function by promoting polarization of microglia toward the M2 type.

Totally 48 SD rats were randomly divided into a sham operation group, a model group, and an exosome group (n=16 per group). A rat spinal cord injury model was established using the modified Allen method. The exosome group was injected with 20 μL of human umbilical cord mesenchymal stem cell-derived exosomes intrathecally via neuroendoscopy 24 hours after injury. At 3, 7, 14, and 21 days after modeling, the recovery of the motor function of the hind limbs of the rats was assessed by BBB scoring method combined with Rivlin's slant plate test. The damage of spinal cord tissues was detected by using hematoxylin-eosin staining and Nissl staining. The expression levels of brain-derived neurotrophic factor and vascular endothelial growth factor A proteins were detected by western blot assay. The expression proportion of M1-type markers (inducible nitric oxide synthase) and M2 markers (arginase-1) in the spinal cord tissues was detected by immunofluorescence method. qRT-PCR and western blot assay were used to detect the expression levels of inducible nitric oxide synthase and arginase-1 in spinal cord tissues. ELISA was utilized to detect the levels of pro-inflammatory factors (tumor necrosis factor α, interleukin 1β, and interleukin 6) and anti-inflammatory factors (interleukin 10) levels in spinal cord tissues.

(1) At 3, 7, and 14 days postoperatively, the BBB scores of the exosome group were better than those of the model group (P < 0.05). The angles of the Rivlin slanting plate experiments of the exosome group were significantly higher than those of the model group at 7 and 14 days postoperatively (P < 0.05). The results of hematoxylin-eosin staining and Nissl staining indicated that the spinal cord tissues and nerve injuries of the exosome group were reduced in comparison with those of the model group, and the levels of brain-derived neurotrophic factor and vascular endothelial growth factor A in spinal cord tissues of the exosome group were higher than those in the model group at 7 days postoperatively (P < 0.05). (2) Immunofluorescence experiments showed that the number of inducible nitric oxide synthase-positive microglial cells in the lesion area of the exosome group was significantly reduced and the level of Arg1-positive microglial cells increased in the lesion area of the exosome group compared with the model group at 7 days postoperatively (P < 0.05). qRT-PCR and western blot assay also confirmed the results of immunofluorescence experiments. (3) The secretion of pro-inflammatory factors tumor necrosis factor α, interleukin 1β, and interleukin 6 in spinal cord tissues of the exosome group was reduced compared with the model group (P < 0.05), whereas the secretion of the inflammation-suppressing factor interleukin 10 was increased compared with the model group (P < 0.05). These findings conclude that human umbilical cord mesenchymal stem cell-derived exosomes could promote the polarization of microglial cells from the M1 to the M2 type and decrease the release of pro-inflammatory factors, thereby reducing the secondary damage of neuroinflammation in spinal cord injury.

Long non-coding RNA (LncRNA) plays an important role in nervous system development and neurological diseases. Previous studies by the research team have demonstrated that human umbilical cord mesenchymal stem cells overexpressing erythropoietin (EPO-MSCs) under ischemic and hypoxic conditions have better neuroprotective functions and significantly activate the expression of LncRNA XIST. Research suggests that XIST is related to the pathogenesis of hypoxic-ischemic encephalopathy, but the role and mechanism of its regulation by EPO-MSCs in protecting ischemic-hypoxic neurons remain unclear.

To explore the new mechanism by which LncRNA XIST, in response to EPO-MSC signaling, affects the apoptosis of ischemic-hypoxic SH-SY5Y cells.

(1) SH-SY5Y cell lines with knockdown of LncRNA XIST (sh-XIST) and negative control (NC-XIST) were constructed through lentiviral transfection. Oxygen-glucose deprivation was used to induce ischemic-hypoxic injury in the cells. Transwell chambers were used to create a non-contact co-culture system with EPO-MSCs, sh-XIST, and NC-XIST ischemic-hypoxic SH-SY5Y cells. Cell proliferation ability was detected using the CCK-8 assay. Cell migration ability was assessed using the scratch assay, and cell apoptosis was measured by flow cytometry. (2) RNA-seq bioinformatics analysis was performed to screen for differentially expressed genes and pathways between sh-XIST and NC-XIST cell lines. Dual-luciferase experiments were used to verify the relationship between miR-124-3p and the target genes XIST and GRIN1. qRT-PCR was conducted to validate the expression levels of downstream miR-124-3p and GRIN1 genes. (3) miR-124-3p inhibitors and mimics were added to verify phenotypic changes in SH-SY5Y cells after ischemic-hypoxic injury and co-culture with EPO-MSCs.

(1) Compared with the NC-XIST group, SH-SY5Y cells in the sh-XIST group showed reduced proliferation and migration abilities and increased apoptosis after ischemic-hypoxic injury and co-culture with EPO-MSCs. (2) Dual-luciferase experiments showed that miR-124-3p interacted with the target gene XIST. SH-SY5Y cells transfected with miR-124-3p mimics and co-cultured with EPO-MSCs showed decreased apoptosis after ischemic-hypoxic injury, while SH-SY5Y cells transfected with miR-124-3p inhibitors showed increased apoptosis after co-culture with EPO-MSCs. (3) Transcriptomic sequencing and bioinformatics analysis of sh-XIST revealed significant downregulation of the neuroactive ligand-receptor pathway and the key receptor gene GRIN1 for central nervous system development. (4) Dual-luciferase experiments showed that miR-124-3p interacted with GRIN1. GRIN1 expression was significantly downregulated in the sh-XIST group after ischemic-hypoxic injury compared with the NC-XIST group. These findings indicate that LncRNA XIST promotes GRIN1 expression by upregulating miR-124-3p, thereby reducing cell apoptosis after ischemic-hypoxic injury and co-culture with EPO-MSCs and enhancing proliferation and migration. sh-XIST can block this protective function.

Erythropoietin/erythropoietin receptor signaling pathway not only participates in bone marrow hematopoiesis, but also regulates the metabolic response of non-hematopoietic tissues, such as brain, heart, skeletal muscle, and adipose tissue. Simultaneously, it can accelerate the mineralization process of periodontal ligament stem cells and reduce oxidative stress damage. However, the mechanism of action on osteogenic differentiation of periodontal ligament stem cells is still unclear.

To investigate the effect and action mechanism of erythropoietin/erythropoietin receptor signaling pathway on osteogenic differentiation of periodontal ligament stem cells.

Enzyme digestion method was used to isolate and culture periodontal ligament stem cells from periodontal disease patients and healthy people. The mRNA and protein levels of erythropoietin receptor in two kinds of periodontal ligament stem cells were detected by qRT-PCR and western blot assay. Erythropoietin receptor expression was silenced by small interfering RNA (siRNA) or activated by erythropoietin. qRT-PCR and western blot assay were used to detect the expression of erythropoietin receptor, levels of osteogenic marker genes Runt-related transcription factor 2 (Runx2), osteocalcin, osteopontin, and bone sialoprotein. Alkaline phosphatase staining and alizarin red staining were applied to measure osteogenic differentiation ability of periodontal ligament stem cells. The phosphorylation of signal transducer and activator of transcription 5 (STAT5) was detected by western blot assay.

(1) The results of qRT-PCR and western blot assay showed that the mRNA and protein levels of erythropoietin receptor in periodontal ligament stem cells in the disease group were significantly lower than those in periodontal ligament stem cells in the healthy group. (2) Alkaline phosphatase staining and alizarin red staining showed that knocking down the erythropoietin receptor can inhibit the osteogenic differentiation ability of periodontal ligament stem cells. qRT-PCR results showed that compared with the control group, knockdown of the erythropoietin receptor group significantly reduced expression levels of Runt-related transcription factor 2, osteocalcin, osteopontin, and bone sialoprotein (P < 0.05). (3) qRT-PCR results showed that after erythropoietin treatment, the expression of erythropoietin receptor in periodontal ligament stem cells recovered. Silencing erythropoietin receptor and then administration of erythropoietin treatment reversed the expression level of erythropoietin receptor. Erythropoietin treatment increased the osteogenic differentiation ability of periodontal ligament stem cells in the disease group and the expression level of the osteogenic marker gene Runt-related transcription factor 2 (P < 0.05). Silencing the expression of STAT5 inhibited this effect of erythropoietin. (4) Western blot assay results showed that with the extension of erythropoietin treatment time, the phosphorylation level of STAT5 increased in periodontal ligament stem cells in the disease group (P < 0.05). The above results indicate that erythropoietin restores the osteogenic differentiation ability of pathological periodontal ligament stem cells by inducing the phosphorylation of STAT5.

Stem cells from human exfoliated deciduous teeth are widely used in the field of tissue repair and regeneration, but the tissue regeneration effect has limitations in practical applications. Using miRNA to intervene in the directional differentiation of stem cells from human exfoliated deciduous teeth is an important development direction for tissue repair and regeneration in the future.

To investigate the effect of miR-26b on the neurogenic and angiogenic differentiation of stem cells from human exfoliated deciduous teeth.

Dental pulp stem cells were isolated and extracted from human exfoliated deciduous teeth, and induced to differentiate into nerves and blood vessels. The expressions of neurogenic markers Nestin, NSE, βIII-Tubulin, and angiogenic markers CD31, VEGFR2, ANG-1 and miR-26b were detected. Dental pulp stem cells were divided into blank control group, miR-26 overexpression group, and negative control group. RT-qPCR, cell immunofluorescence staining, and western blot assay were used to detect the expression changes of related markers of neurogenic and angiogenic induction of stem cells from human exfoliated deciduous teeth in each group.

(1) Stem cells from human exfoliated deciduous teeth had multi-directional differentiation potential and could differentiate into osteogenesis, adipogenesis, neurogenesis, and vasculogenesis. (2) The expression level of miR-26b increased during the neurogenic and angiogenic differentiation of stem cells from human exfoliated deciduous teeth. (3) Compared with the blank control group and negative control group, the mRNA expression of neuroblast-related genes βIII-Tubulin, Nestin, NSE and angiogenesis-related genes CD31, VEGFR2, and ANG-1 in stem cells from human exfoliated deciduous teeth in the miR-26b overexpression group was significantly increased (P < 0.01), βIII-Tubulin, Nestin, CD31, and VEGFR2 protein expression was significantly increased (P < 0.01). The above results show that overexpression of miR-26b can promote the neurogenic and angiogenic differentiation of stem cells from human exfoliated deciduous teeth.

Long noncoding RNA TP53TG1 (lncRNA TP53TG1) is involved in regulating the proliferation, migration, invasion, and apoptosis of various cancer cells, but there are few reports on its role in other cells.

To investigate the effects and pathways of lncRNA TP53TG1 on the proliferation and differentiation of human stem cells from the apical papilla.

Human stem cells from the apical papilla were isolated and cultured, and then transfected with lncRNA TP53TG1 overexpression lentivirus. RT-qPCR was used to detect the overexpression efficiency of lncRNA TP53TG1. Western blot assay was used to detect the relative expression levels of PI3K, AKT, ERK, P38, Smad3, and their phosphorylated proteins. Human stem cells from the apical papilla were divided into the empty lentiviral vector transfection group and the lncRNA TP53TG1 overexpression group. CCK-8 assay was used to measure the cell proliferation. Alkaline phosphatase activity was detected by alkaline phosphatase staining on day 5 of osteogenic induction. Formation of mineralized nodules was detected by alizarin red staining on day 21 of osteogenic induction. RT-qPCR was used to detect the mRNA expression levels of dentin sialophosphoprotein, Runt-related transcription factor 2, dentin matrix protein 1, and bone sialoprotein on days 3, 7, and 14 of osteogenic induction. Western blot assay was used to detect the protein expression levels of dentin sialophosphoprotein and Runt-related transcription factor 2 on days 3, 7, and 14 of osteogenic induction.

(1) RT-qPCR results showed that the lentivirus was successfully integrated into the genome of stem cells from the apical papilla. Western blot assay results showed that overexpression of lncRNA TP53TG1 up-regulated the protein levels of p-PI3K and p-AKT without affecting the expression of phosphorylated proteins in other pathways. (2) Starting from day 3 of cell culture, overexpression of lncRNA TP53TG1 significantly promoted the proliferation of stem cells from the apical papilla. (3) In the process of inducing odontogenic differentiation of stem cells from the apical papilla, overexpression of lncRNA TP53TG1 promoted the expression of odontogenic and osteogenic differentiation-related genes and proteins, significantly increased alkaline phosphatase activity and mineralized nodule formation. (4) The results show that lncRNA TP53TG1 may promote the odontogenic and osteogenic differentiation of stem cells from the apical papilla by activating the PI3K/AKT signaling pathway.