The cryopreservation technology enables tissues/cells to be stored for a long time in a low-temperature environment while maintaining the integrity of their activity and function, which is of great significance for the construction of cell therapy, tissue engineering and biological sample banks. Cryoprotective agents often contain dimethyl sulfoxide and serum. To avoid the toxic side effects of dimethyl sulfoxide, the complexity of serum components and immune responses, although some finished cryoprotective agents have been marketed, they are faced with many difficulties such as high cost and limited application. Therefore, there is an urgent need to develop a cryoprotective agent with clear components and the ability to solve the above problems.

To evaluate the effects of a novel cryoprotectant on cryopreservation efficiency of different tissue and cell sources.

By applying the novel cryoprotectant as an experimental group with the commercially available and widely used cryoprotectant (control group) to umbilical cord Wharton's jelly tissue, umbilical cord mesenchymal stem cells, umbilical cord blood/peripheral blood mononuclear cells, NK and CIK cells, comparative analyses were conducted in terms of cell morphology, number, viability, surface markers, differentiation potential, and cell-killing toxicity assay before cryopreservation and after resuscitation thawing. We confirmed the cryopreservation effect of the new cryoprotectant and its potential application value.

(1) The novel cryoprotectant facilitated the normal growth of cryopreserved Wharton's jelly tissue upon recovery, exhibiting mesenchymal stem cell morphology. No significant differences were observed between the experimental and control groups in terms of cell recovery rate, surface markers, and differentiation potential. (2) There was no significant difference in the number and viability of cells between the experimental group and the control group after cryopreservation of cord blood/peripheral blood mononuclear cells, and the cryo-resuscitated cell numbers and viability of derived NK cells/CIK cells did not show significant difference between the experimental and control groups. (3) For NK cells derived and differentiated from cord blood/peripheral blood mononuclear cells, there was no significant difference in the proportion of CD56⁺CD16⁺ cell subpopulations between the experimental group and the control group. For CIK cells derived and differentiated from cord blood/peripheral blood mononuclear cells, there was no significant difference in the proportions of CD3⁺CD8⁺ and CD3⁺CD56⁺ cell subpopulations between the experimental group and the control group. (4) In terms of cytotoxicity testing, when the effective-target ratio of immune cells and melanoma cell line Mel624 was 20:1, whether it was NK cells/CIK cells derived from cord blood or peripheral blood mononuclear cells, there was no significant difference in the tumoricidal activity of cells between the experimental group and the control group. These findings suggest that the novel cryoprotectant can replace existing commercially available and widely used cryoprotectants, and is applicable to Wharton's jelly tissue, umbilical cord mesenchymal stem cells, umbilical cord blood/peripheral blood mononuclear cells, as well as NK and CIK cells, providing a solid technical foundation for the scaling, standardization, and commercialization of universal cryoprotectants.

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.

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.

Radiotherapy for head and neck tumors can easily cause xerostomia, seriously affecting the quality of life of patients. In recent years, engineered stem cells and their paracrine factors have shown therapeutic potential in the repair of salivary gland injury. However, there is currently no experimental study on the application of amniotic mesenchymal stem cell-derived exosome in radiation-induced salivary gland injury.

To preliminarily explore the repair effect of exosome derived from human amniotic mesenchymal stem cells on radiation-induced submandibular gland injury.

Human amniotic mesenchymal stem cell exosomes were extracted and identified by ultrafiltration and ultracentrifugation. SD rats were randomly divided into a control group, a radiation injury group, and a radiation injury+exosome group. An in vitro model of radiation-induced submandibular gland injury was constructed using the submandibular gland tissue of SD rats irradiated with 18 Gy of radiation. One day after radiation modeling, exosome derived from human amniotic mesenchymal stem cells was injected into the submandibular gland in situ. Samples are taken at 1, 3, 7, and 14 days to detect the resting salivary flow rate. The structure of the submandibular gland tissue was observed by hematoxylin-eosin staining. The expression of glycogen particles in the submandibular gland tissue was observed by Periodic Acid-Schiff staining. Fibrosis in the submandibular gland tissue was observed by Masson staining. The secretion of salivary amylase was detected by enzyme-linked immunosorbent assay. The expression of aquaporin and tight junction proteins in submandibular gland tissue was observed by immunofluorescence staining. Real-time fluorescence quantitative PCR was used to detect the relative expression levels of aquaporins and salivary amylase mRNA in submandibular gland tissue. TUNEL assay was used to detect the apoptosis rate of submandibular gland tissues in each group.

After radiomodeling, compared with the radiation injury group, (1) hematoxylin-eosin staining observed that the submandibular gland tissue structure in the radiation injury+exosome group was restored, the nucleoli increased, the number of acinus increased, and the acinar atrophy improved. (2) Glycogen staining observed that the number and density of positive zymogen granules in the acinar cytoplasm of the radiation injury+exosome group gradually increased. (3) Masson staining results observed that the number and density of positive collagen fibers in the interstitium and around the ducts in the radiation injury+exosome group gradually decreased, the degree of fibrosis decreased, and the collagen deposition decreased. (4) The salivary flow rate in the radiation injury+exosome group increased (P < 0.05). The fluorescence intensity of aquaporin-5 was enhanced (P < 0.05) and the gene expression was significantly enhanced (P < 0.01). The fluorescence distribution of tight junction protein 4 was weakened and the fluorescence intensity decreased (P < 0.05, P < 0.01). The content of salivary amylase increased (P < 0.05) and gene expression were significantly increased (P < 0.01). The number of positive apoptotic cells decreased (P < 0.05, P < 0.01). It is indicated that local injection of exosome derived from human amniotic mesenchymal stem cells could improve the pathological morphology of submandibular gland tissue, promote saliva flow rate and amylase expression, and may play a functional repair role in radioactive submandibular gland injury by inhibiting acinar apoptosis.

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.

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.

Numerous studies have indicated that pyroptosis plays a key role in the progression of cancer. In recent years, research has shown that pyroptosis is inextricably linked to the occurrence, development, and treatment of breast cancer. The development of effective pyroptosis-based therapeutic strategies has become a hot topic in the field of breast cancer treatment.

To comprehensively analyze the mechanisms of pyroptosis, explore the role of pyroptosis in the anti-tumor effects in breast cancer, and its potential application value in clinical treatment.

Using English search terms “pyroptosis, breast cancer, inflammasome, gasdermin, caspase, drug resistance, treatment”, PubMed database was searched for articles published from inception to August 2024. Through the preliminary screening of reading titles and abstracts, literature with poor relevance to the research content, outdated information, repeated views, and lack of authority was excluded. Finally, 121 articles were included for review.

Pyroptosis is a special form of programmed cell death that is carried out by the activation of the gasdermin family of proteins, showing potential application value in the treatment of breast cancer. Long-term or improper treatment can lead to drug resistance in cancer cells; research on the mechanism of pyroptosis helps to overcome resistance deficiencies. Pyroptosis can trigger immunogenic cell death, promoting the release of tumor-specific antigens, thereby activating the immune system and enhancing its ability to recognize and clear tumor cells. The expression levels of pyroptosis-related genes can serve as prognostic indicators for breast cancer, helping to assess patients’ treatment responses and survival periods. Research on the mechanisms of pyroptosis can provide new strategies for the treatment of breast cancer, such as targeted drugs and therapeutic methods that induce pyroptosis, contributing to the realization of personalized treatment plans for breast cancer.

Human pluripotent stem cell-derived cardiomyocytes offer an ideal cellular resource for studying heart diseases, conducting drug screening, developing in vitro heart models, and exploring potential cell therapies. However, human pluripotent stem cell-derived cardiomyocytes are characterized by immaturity with limited specific gene expression, low Ca²⁺ processing levels, and underdeveloped structural, metabolic, and electrophysiological features. These limitations significantly impede the application of human pluripotent stem cell-derived cardiomyocytes.

To review the academic progress and clinical application of promoting the maturation of human pluripotent stem cell-derived cardiomyocytes by in vitro synthetic microenvironment.

CNKI, WanFang, VIP, PubMed, Web of Science, and Medline databases were searched, with “human pluripotent stem cells, human myocardial cells, hPSC-CMs, mature, OA, human pluripotent stem cell-derived cardiomyocytes, hPSC-CMs” as English search terms and “human pluripotent stem cells, cardiomyocytes, mature, OA, hPSC-CMs” as Chinese search terms. All relevant literature published from January 2002 to July 2024 was retrieved and 82 articles were included in the review.

(1) In recent years, in vitro synthetic microenvironments have attracted extensive attention due to their excellent intrinsic properties such as stiffness, plasticity, nanoscale morphology, and chemical functionality. (2) Human pluripotent stem cell-derived cardiomyocytes can be used as an effective platform for the treatment of cardiovascular diseases. (3) Mechanical stimulation, electrical stimulation, addition of biochemical molecules, and three-dimensional culture methods are effective methods to promote the maturation of human pluripotent stem cell-derived cardiomyocytes, which can further promote the clinical application of human pluripotent stem cell-derived cardiomyocytes.

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.

Extracellular vesicles have received extensive attention in the field of bone defect regeneration and repair in recent years. However, natural extracellular vesicles have deficiencies in sustained controlled release, tissue targeting, and drug loading capacity. Therefore, the introduction of engineering strategies to modify extracellular vesicles to enhance their therapeutic efficacy has become a research hotspot.

To review the role and application progress of engineered extracellular vesicles in the regeneration and repair of bone defects.

PubMed, Web of Science, CNKI, and WanFang databases were searched for relevant articles published in the past fifteen years. The search terms were “engineering, extracellular vesicles, exosomes, bone defect, bone regeneration, bone repair” in Chinese and English. After removal of poorly related, outdated, and duplicate studies by screening, 93 articles were finally included for review according to inclusion criteria.

(1) Extracellular vesicles are primarily isolated based on their density, size, immunoaffinity, and surface charge. After isolation, extracellular vesicles are characterized using imaging techniques, size- and counting-based techniques, and flow cytometry. (2) Extracellular vesicles stimulate bone regeneration by regulating immunity, angiogenesis, and proliferation and differentiation of target cells. (3) The engineering strategies of extracellular vesicles include surface modification and cargo loading. (4) The introduction of bone morphogenetic protein 2, mutant hypoxia-inducible factor-1α, vascular endothelial growth factor, miRNA and other bioactive factors into extracellular vesicles through engineering strategies can enhance their regenerative repair ability for bone defects.