Therapeutic potential of stem cell and melatonin on the reduction of CCl4-induced liver fibrosis in experimental mice model / Potencial terapêutico de células-tronco e melatonina na redução da fibrose hepática induzida por CCl4 no modelo de ratos experimentais

Liver fibrosis is initial stage of any chronic liver disease and its end stage is develops into cirrhosis. Chronic liver diseases are a crucial global health issue and the cause of approximately 2 million deaths per year worldwide. Cirrhosis is currently the 11th most common cause of death globally. Mesenchymal stem cell (MSCs) treatment is the best way to treat acute and chronic liver disease. The aim of this study is to improve the therapeutic potential of MSCs combined with melatonin (MLT) to overcome CCl4-induced liver fibrosis and also investigate the individual impact of melatonin and MSCs against CCl4-induced liver impairment in animal model. Female BALB/c mice were used as CCL4-induced liver fibrotic animal model. Five groups of animal model were made; negative control, Positive control, CCl4+MSCs treated group, CCl4+MLT treated group and CCl4+MSCs+MLT treated group. Cultured MSCs from mice bone marrow were transplanted to CCl4-induced liver injured mice model, individually as well as together with melatonin. Two weeks after MSCs and MLT administration, all groups of mice were sacrificed for examination. Morphological and Histopathological results showed that combined therapy of MSCs+MLT showed substantial beneficial impact on CCl4-induced liver injured model, compared with MSCs and MLT individually. Biochemically, considerable reduction was observed in serum bilirubin and ALT levels of MLT+MSC treated mice, compared to other groups. PCR results shown down-regulation of Bax and up-regulation of Bcl-xl and Albumin, confirm a significant therapeutic effect of MSCs+MLT on CCI4-induced liver fibrosis. From the results, it is concluded that combined therapy of MSCs and MLT show strong therapeutic effect on CCL4-induced liver fibrosis, compared with MSCs and MLT individually.

A fibrose hepática é a fase inicial de qualquer doença hepática crônica, e em sua fase final desenvolve-se para cirrose. As doenças hepáticas crônicas são uma questão de saúde global crucial e a causa de aproximadamente 2 milhões de mortes por ano em todo o mundo. A cirrose, hoje em dia, é a 11ª causa mais comum de morte globalmente. O tratamento da célula-tronco mesenquimal (MSCs) é uma maneira eletiva de tratar a doença hepática aguda e crônica. O objetivo deste estudo é melhorar o potencial terapêutico dos MSCs combinados com a melatonina (MLT) para superar a fibrose hepática induzida por CCl4 e também investigar o impacto individual da melatonina e MSCs contra o comprometimento do fígado induzido por CCl4 no modelo animal. Os ratos BALB / C fêmeas foram usados ​​como modelo de animal fibrótico de fígado induzido por CCl4. Cinco grupos de modelo animal foram feitos: Controle Negativo, Controle Positivo, CCl4 + MSCs Tratados Grupo, Grupo Tratado CCl4 + MLT e Grupo Tratado CCl4 + MSCs + MLT. MSCs cultivados da medula óssea dos ratos foram transplantados para o modelo de camundongos de fígado induzido por CCl4, individualmente, bem como em conjunto com a melatonina. Duas semanas após a administração MSCs e MLT, todos os grupos de camundongos foram sacrificados para o exame. Os resultados morfológicos e histopatológicos mostraram que a terapia combinada do MSCs + MLT mostrou impacto benéfico substancial no modelo ferido no fígado induzido pelo CCl4, em comparação com o MSCs e o MLT individualmente. A redução bioquimicamente considerável foi observada em bilirrubina sérica e níveis ALT de ratinhos tratados com MLT + MSCs, em comparação com outros grupos. Os resultados de PCR mostraram regulação negativa do BAX e regulação positiva do BCL-XL e da albumina, confirmando um efeito terapêutico significativo do MSCs + MLT na fibrose hepática induzida por CCl4. Dos resultados, conclui-se que a terapia combinada de MSCs e MLT mostram um forte efeito terapêutico na fibrose hepática induzida por CCl4, em comparação com MSCs e MLT individualmente.

Potential effect of platelet rich fibrin prepared under differen centrifugation protocols on stem cells from the apical papila / O potencial efeito da fibrina rica em plaquetas preparada sob diferentes protocolos de centrifugação nas células tronco da papila apical

Objetivo: Este estudo objetivou avaliar o potencial proliferativo e de diferenciação das células tronco da papila cultivadas conjuntamente com fibrina rica em plaquetas (PRF) preparados sob dois protocolos de centrifugação distintos. Material e Métodos: Protocolos padrão e avançado de PRF foram utilizados. As células foram divididas em 4 grupos: controle negativo, controle positivo, padrão (L-PRF) e avançado (A-PRF). A contagem de células e ensaio de viabilidade foram realizados para verificar a capacidade proliferativa. Coloração vermelho de alizarina S, atividade de fosfatase alcalina e imunofluorescência para o receptor ativador do fator nuclear kappa-B (RANKL) foram utilizados para avaliar o potencial osteogênico e de diferenciação celular. Resultados: Ambos os tipos de PRF aumentaram o número de células, viabilidade celular sem toxicidade o que refletiu no aumento da proliferação e diferenciação de acordo com os testes realizados. Conclusão: O grupo A-PRF aumentou significativamente a proliferação e diferenciação comparado com o grupo L-PRF.(AU)

Objectives: The present work was designed to evaluate the proliferation and differentiation potential of stem cells from the apical papilla (SCAP) seeded along with platelet rich fibrin (PRF) scaffolds prepared under two different centrifugation protocols. Materials and Methods: Standard and advanced PRF protocols were used. Cells were divided into 4 groups: negative control, positive control, standard (L-PRF) and advanced (A-PRF) groups. Cell count and cell viability assays were carried out to assess the proliferation capacity. Alizarin red S (ARS) stain, Alkaline phosphatase (ALP) activity and Receptor activator of nuclear factor-kappa B ligand (RANKL) immunofluorescence staining were used to evaluate the osteogenic potential in the differentiated cells. Results:Both types of platelet rich fibrin increased the cell count, cell viability with no cytotoxicity that was reflected on increased proliferation and differentiation in terms of the performed tests. Conclusion: A-PRF group showed significant increase in proliferation and differentiation potentials compared to L-PRF group

Stem cell therapy in stargardt disease: a systematic review

This article aimed to review current literature on the safety and efficacy of stem cell therapy in Stargardt disease. A comprehensive literature search was performed, and two animal and eleven human clinical trials were retrieved. These studies utilized different kinds of stem cells, including human or mouse embryonic stem cells, mesenchymal stem cells, bone marrow mononuclear fraction, and autologous bone marrow-derived stem cells. In addition, different injection techniques including subretinal, intravitreal, and suprachoroidal space injections have been evaluated. Although stem cell therapy holds promise in improving visual function in patients with Stargardt disease, further investigation is needed to determine the long-term benefits, safety, and efficacy in determining the best delivery method and selecting the most appropriate stem cell type.

Study of the inflammatory activating process in the early stage of Fusobacterium nucleatum infected PDLSCs / 国际口腔科学杂志·英文版

Fusobacterium nucleatum (F. nucleatum) is an early pathogenic colonizer in periodontitis, but the host response to infection with this pathogen remains unclear. In this study, we built an F. nucleatum infectious model with human periodontal ligament stem cells (PDLSCs) and showed that F. nucleatum could inhibit proliferation, and facilitate apoptosis, ferroptosis, and inflammatory cytokine production in a dose-dependent manner. The F. nucleatum adhesin FadA acted as a proinflammatory virulence factor and increased the expression of interleukin(IL)-1β, IL-6 and IL-8. Further study showed that FadA could bind with PEBP1 to activate the Raf1-MAPK and IKK-NF-κB signaling pathways. Time-course RNA-sequencing analyses showed the cascade of gene activation process in PDLSCs with increasing durations of F. nucleatum infection. NFκB1 and NFκB2 upregulated after 3 h of F. nucleatum-infection, and the inflammatory-related genes in the NF-κB signaling pathway were serially elevated with time. Using computational drug repositioning analysis, we predicted and validated that two potential drugs (piperlongumine and fisetin) could attenuate the negative effects of F. nucleatum-infection. Collectively, this study unveils the potential pathogenic mechanisms of F. nucleatum and the host inflammatory response at the early stage of F. nucleatum infection.

Effect of naringenin on the anti-inflammatory, vascularization, and osteogenesis differentiation of human periodontal ligament stem cells via the stromal cell-derived factor 1/C-X-C motif chemokine receptor 4 signaling axis stimulated by lipopolysaccharide / 华西口腔医学杂志

OBJECTIVES@#This study aimed to investigate how naringenin (Nar) affected the anti-inflammatory, vascula-rization, and osteogenesis differentiation of human periodontal ligament stem cells (hPDLSCs) stimulated by lipopolysaccharide (LPS) and to preliminarily explore the underlying mechanism.@*METHODS@#Cell-counting kit-8 (CCK8), cell scratch test, and Transwell assay were used to investigate the proliferation and migratory capabilities of hPDLSCs. Alkaline phosphatase (ALP) staining, alizarin red staining, lumen-formation assay, enzyme-linked immunosorbent assay, quantitative timed polymerase chain reaction, and Western blot were used to measure the expression of osteopontin (OPN), Runt-related transcription factor 2 (RUNX2), vascular endothlial growth factor (VEGF), basic fibroblast growth factor (bFGF), von Willebrand factor (vWF), tumor necrosis factor-α (TNF-α), and interleukin (IL)-6.@*RESULTS@#We observed that 10 μmol/L Nar could attenuate the inflammatory response of hPDLSCs stimulated by 10 μg/mL LPS and promoted their proliferation, migration, and vascularization differentiation. Furthermore, 0.1 μmol/L Nar could effectively restore the osteogenic differentiation of inflammatory hPDLSCs. The effects of Nar's anti-inflammatory and promotion of osteogenic differentiation significantly decreased and inflammatory vascularization differentiation increased after adding AMD3100 (a specific CXCR4 inhibitor).@*CONCLUSIONS@#Nar demonstrated the ability to promote the anti-inflammatory, vascularization, and osteogenic effects of hPDLSCs stimulated by LPS, and the ability was associated with the stromal cell-derived factor/C-X-C motif chemokine receptor 4 signaling axis.

Spontaneous spheroids from alveolar bone-derived mesenchymal stromal cells maintain pluripotency of stem cells by regulating hypoxia-inducible factors

BACKGROUND: Spontaneous spheroid culture is a novel three-dimensional (3D) culture strategy for the rapid and efficient selection of progenitor cells. The objectives of this study are to investigate the pluripotency and differentiation capability of spontaneous spheroids from alveolar bone-derived mesenchymal stromal cells (AB-MSCs); compare the advantages of spontaneous spheroids to those of mechanical spheroids; and explore the mechanisms of stemness enhancement during spheroid formation from two-dimensional (2D) cultured cells. METHODS: AB-MSCs were isolated from the alveolar bones of C57BL/6 J mice. Spontaneous spheroids formed in low-adherence specific culture plates. The stemness, proliferation, and multi-differentiation capacities of spheroids and monolayer cultures were investigated by reverse transcription quantitative polymerase chain reaction (RT-qPCR), immunofluorescence, alkaline phosphatase (ALP) activity, and oil-red O staining. The pluripotency difference between the spontaneous and mechanical spheroids was analyzed using RT-qPCR. Hypoxia-inducible factor (HIFs) inhibition experiments were performed to explore the mechanisms of stemness maintenance in AB-MSC spheroids. RESULTS: AB-MSCs successfully formed spontaneous spheroids after 24 h. AB-MSC spheroids were positive for MSC markers and pluripotency markers (Oct4, KLF4, Sox2, and cMyc). Spheroids showed higher Ki67 expression and lower Caspase3 expression at 24 h. Under the corresponding conditions, the spheroids were successfully differentiated into osteogenic and adipogenic lineages. AB-MSC spheroids can induce neural-like cells after neurogenic differentiation. Higher expression of osteogenic markers, adipogenic markers, and neurogenic markers (NF-M, NeuN, and GFAP) was found in spheroids than in the monolayer. Spontaneous spheroids exhibited higher stemness than mechanical spheroids did. HIF-1α and HIF-2α were remarkably upregulated in spheroids. After HIF-1/2α-specific inhibition, spheroid formation was significantly reduced. Moreover, the expression of the pluripotency genes was suppressed. CONCLUSIONS: Spontaneous spheroids from AB-MSCs enhance stemness and pluripotency. HIF-1/2α plays an important role in the stemness regulation of spheroids. AB-MSC spheroids exhibit excellent multi-differentiation capability, which may be a potent therapy for craniomaxillofacial tissue regeneration.

Isolation, culture and validation of CD34+ vascular wall-resident stem cells from mice / 生理学报

Vascular wall-resident stem cells (VW-SCs) play a critical role in maintaining normal vascular function and regulating vascular repair. Understanding the basic functional characteristics of the VW-SCs will facilitate the study of their regulation and potential therapeutic applications. The aim of this study was to establish a stable method for the isolation, culture, and validation of the CD34+ VW-SCs from mice, and to provide abundant and reliable cell sources for further study of the mechanisms involved in proliferation, migration and differentiation of the VW-SCs under various physiological and pathological conditions. The vascular wall cells of mouse aortic adventitia and mesenteric artery were obtained by the method of tissue block attachment and purified by magnetic microbead sorting and flow cytometry to obtain the CD34+ VW-SCs. Cell immunofluorescence staining was performed to detect the stem cell markers (CD34, Flk-1, c-kit, Sca-1), smooth muscle markers (SM22, SM MHC), endothelial marker (CD31), and intranuclear division proliferation-related protein (Ki-67). To verify the multipotency of the isolated CD34+ VW-SCs, endothelial differentiation medium EBM-2 and fibroblast differentiation medium FM-2 were used. After culture for 7 days and 3 days respectively, endothelial cell markers and fibroblast markers of the differentiated cells were evaluated by immunofluorescence staining and q-PCR. Furthermore, the intracellular Ca2+ release and extracellular Ca2+ entry signaling were evaluated by TILLvisION system in Fura-2/AM loaded cells. The results showed that: (1) High purity (more than 90%) CD34+ VW-SCs from aortic adventitia and mesenteric artery of mice were harvested by means of tissue block attachment method and magnetic microbead sorting; (2) CD34+ VW-SCs were able to differentiate into endothelial cells and fibroblasts in vitro; (3) Caffeine and ATP significantly activated intracellular Ca2+ release from endoplasmic reticulum of CD34+ VW-SCs. Store-operated Ca2+ entry (SOCE) was activated by using thapsigargin (TG) applied in Ca2+-free/Ca2+ reintroduction protocol. This study successfully established a stable and efficient method for isolation, culture and validation of the CD34+ VW-SCs from mice, which provides an ideal VW-SCs sources for the further study of cardiovascular diseases.

Research progress in lineage tracing to explore hepatic parenchymal cell regeneration and repair mechanisms / 中华肝脏病杂志

Hepatic parenchymal cells are a type of liver cells that performs important functions such as metabolism and detoxification. The contribution of hepatic parenchymal cells, bile duct cells, and hepatic stem/progenitor cells to new hepatic parenchymal cells in the process of liver injury repair has become a controversial issue due to their strong proliferation ability. Lineage tracing technology, which has emerged in the past decade as a new method for exploring the origin of cells, can trace specific type of cells and their daughter cells by labeling cells that express the specific gene and their progeny. The article reviews the current literature on the origin and contribution of hepatic parenchymal cells by this technique. About 98% of new hepatic parenchymal cells originate from the existing hepatic parenchymal cells during liver homeostasis and after acute injury. However, under conditions of severe liver injury, such as inhibition of hepatic parenchymal cell proliferation, bile duct cells (mainly liver stem/progenitor cells) become the predominant source of hepatic parenchymal cells, contributing a steady increased hepatocyte regeneration with the extension of time.

Influence of autologous adipose stem cell matrix gel on wound healing and scar hyperplasia of full-thickness skin defects in rabbit ears / 中华烧伤杂志

Objective: To investigate the influence of autologous adipose stem cell matrix gel on wound healing and scar hyperplasia of full-thickness skin defects in rabbit ears, and to analyze the related mechanism. Methods: Experimental research methods were adopted. The complete fat pads on the back of 42 male New Zealand white rabbits aged 2 to 3 months were cut to prepare adipose stem cell matrix gel, and a full-thickness skin defect wound was established on the ventral side of each ear of each rabbit. The left ear wounds were included in adipose stem cell matrix gel group (hereinafter referred to as matrix gel group), and the right ear wounds were included in phosphate buffer solution (PBS) group, which were injected with autologous adipose stem cell matrix gel and PBS, respectively. The wound healing rate was calculated on post injury day (PID) 7, 14, and 21, and the Vancouver scar scale (VSS) scoring of scar tissue formed on the wound (hereinafter referred to as scar tissue) was performed in post wound healing month (PWHM) 1, 2, 3, and 4. Hematoxylin-eosin staining was performed to observe and measure the histopathological changes of wound on PID 7, 14, and 21 and the dermal thickness of scar tissue in PWHM 1, 2, 3, and 4. Masson staining was performed to observe the collagen distribution in wound tissue on PID 7, 14, and 21 and scar tissue in PWHM 1, 2, 3, and 4, and the collagen volume fraction (CVF) was calculated. The microvessel count (MVC) in wound tissue on PID 7, 14, and 21 and the expressions of transforming growth factor β1 (TGF-β1) and α smooth muscle actin (α-SMA) in scar tissue in PWHM 1, 2, 3, and 4 were detected by immunohistochemical method, and the correlation between the expression of α-SMA and that of TGF-β1 in scar tissue in matrix gel group was analyzed. The expressions of vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) in wound tissue were detected by enzyme-linked immunosorbent assay on PID 7, 14, and 21. The number of samples at each time point in each group was 6. Data were statistically analyzed with analysis of variance for repeated measurement, analysis of variance for factorial design, paired sample t test, least significant difference test, and Pearson correlation analysis. Results: On PID 7, the wound healing rate in matrix gel group was (10.3±1.7)%, which was close to (8.5±2.1)% in PBS group (P>0.05). On PID 14 and 21, the wound healing rates in matrix gel group were (75.5±7.0)% and (98.7±0.8)%, respectively, which were significantly higher than (52.7±6.7)% and (90.5±1.7)% in PBS group (with t values of 5.79 and 10.37, respectively, P<0.05). In PWHM 1, 2, 3, and 4, the VSS score of scar tissue in matrix gel group was significantly lower than that in PBS group (with t values of -5.00, -2.86, -3.31, and -4.45, respectively, P<0.05). Compared with the previous time point within the group, the VSS score of scar tissue at each time point after wound healing in the two groups was significantly increased (P<0.05), except for PWHM 4 in matrix gel group (P>0.05). On PID 7, the granulation tissue regeneration and epithelialization degree of the wounds between the two groups were similar. On PID 14 and 21, the numbers of fibroblasts, capillaries, and epithelial cell layers in wound tissue of matrix gel group were significantly more than those in PBS group. In PWHM 1, 2, 3, and 4, the dermal thickness of scar tissue in matrix gel group was significantly thinner than that in PBS group (with t values of -4.08, -5.52, -6.18, and -6.30, respectively, P<0.05). Compared with the previous time point within the group, the dermal thickness of scar tissue in the two groups thickened significantly at each time point after wound healing (P<0.05). Compared with those in PBS group, the collagen distribution in wound tissue in matrix gel group was more regular and the CVF was significantly increased on PID 14 and 21 (with t values of 3.98 and 3.19, respectively, P<0.05), and the collagen distribution in scar tissue was also more regular in PWHM 1, 2, 3, and 4, but the CVF was significantly decreased (with t values of -7.38, -4.20, -4.10, and -4.65, respectively, P<0.05). Compared with the previous time point within the group, the CVFs in wound tissue at each time point after injury and scar tissue at each time point after wound healing in the two groups were significantly increased (P<0.05), except for PWHM 1 in matrix gel group (P>0.05). On PID 14 and 21, the MVC in wound tissue in matrix gel group was significantly higher than that in PBS group (with t values of 4.33 and 10.10, respectively, P<0.05). Compared with the previous time point within the group, the MVC of wound at each time point after injury in the two groups was increased significantly (P<0.05), except for PID 21 in PBS group (P>0.05). In PWHM 1, 2, 3, and 4, the expressions of TGF-β1 and α-SMA in scar tissue in matrix gel group were significantly lower than those in PBS group (with t values of -2.83, -5.46, -5.61, -8.63, -10.11, -5.79, -8.08, and -11.96, respectively, P<0.05). Compared with the previous time point within the group, the expressions of TGF-β1 and α-SMA in scar tissue in the two groups were increased significantly at each time point after wound healing (P<0.05), except for the α-SMA expression in matrix gel group in PWHM 4 (P>0.05). There was a significantly positive correlation between the expression of α-SMA and that of TGF-β1 in scar tissue in matrix gel group (r=0.92, P<0.05). On PID 14 and 21, the expressions of VEGF (with t values of 6.14 and 6.75, respectively, P<0.05) and EGF (with t values of 8.17 and 5.85, respectively, P<0.05) in wound tissue in matrix gel group were significantly higher than those in PBS group. Compared with the previous time point within the group, the expression of VEGF of wound at each time point after injury in the two groups was increased significantly (P<0.05), and the expression of EGF was decreased significantly (P<0.05). Conclusions: Adipose stem cell matrix gel may significantly promote the wound healing of full-thickness skin defects in rabbit ears by promoting collagen deposition and expressions of VEGF and EGF in wound tissue, and may further inhibit the scar hyperplasia after wound healing by inhibiting collagen deposition and expressions of TGF-β1 and α-SMA in scar tissue.

Research advances of adipose stem cell matrix gel in promoting wound healing / 中华烧伤杂志

In recent years, with the problem of aging population in China being prominant, the number of patients with chronic wounds such as diabetic foot, pressure ulcer, and vascular ulcer is increasing. Those diseases seriously affect the life quality of patients and increase the economy and care burden of the patients' family, which have been one of the most urgent clinical problems. Many researches have confirmed that adipose stem cells can effectively promote wound healing, while exogenous protease is needed, and there are ethical and many other problems, which limit the clinical application of adipose stem cells. Adipose stem cell matrix gel is a gel-like mixture of biologically active extracellular matrix and stromal vascular fragment obtained from adipose tissue by the principle of fluid whirlpool and flocculation precipitation. It contains rich adipose stem cells, hematopoietic stem cells, endothelial progenitor cells, and macrophages, etc. The preparation method of adipose stem cell matrix gel is simple and the preparation time is short, which is convenient for clinical application. Many studies at home and abroad showed that adipose stem cell matrix gel can effectively promote wound healing by regulating inflammatory reaction, promoting microvascular reconstruction and collagen synthesis. Therefore, this paper summarized the preparation of adipose stem cell matrix gel, the mechanism and problems of the matrix gel in promoting wound repair, in order to provide new methods and ideas for the treatment of chronic refractory wounds in clinic.

Establishment and Evaluation of Intestinal Injury Model of Mouse Acute Graft Versus Host Disease Based on An Organoid Technology / 中国实验血液学杂志

OBJECTIVE@#To establish an intestinal organoid model that mimic acute graft versus host disease (aGVHD) caused intestinal injuries by using aGVHD murine model serum and organoid culture system, and explore the changes of aGVHD intestine in vitro by advantage of organoid technology.@*METHODS@#20-22 g female C57BL/6 mice and 20-22 g female BALB/c mice were used as donors and recipients for bone marrow transplantation, respectively. Within 4-6 h after receiving a lethal dose (8.0 Gy) of γ ray total body irradiation, a total of 0.25 ml of murine derived bone marrow cells (1×107/mice, n=20) and spleen nucleated cells (5×106/mice, n=20) was infused to establish a mouse model of aGVHD (n=20). The aGVHD mice were anesthetized at the 7th day after transplantation, and the veinal blood was harvested by removing the eyeballs, and the serum was collected by centrifugation. The small intestinal crypts of healthy C57BL/6 mice were harvested and cultivated in 3D culture system that maintaining the growth and proliferation of intestinal stem cells in vitro. In our experiment, 5%, 10%, 20% proportions of aGVHD serum were respectively added into the organoid culture system for 3 days. The formation of small intestinal organoids were observed under an inverted microscope and the morphological characteristics of intestinal organoids in each groups were analyzed. For further evaluation, the aGVHD intestinal organoids were harvested and their pathological changes were observed. Combined with HE staining, intestinal organ morphology evaluation was performed. Combined with Alcian Blue staining, the secretion function of aGVHD intestinal organoids was observed. The distribution and changes of Lgr5+ and Clu+ intestinal stem cells in intestinal organoids were analyzed under the conditions of 5%, 10% and 20% serum concentrations by immunohistochemical stainings.@*RESULTS@#The results of HE staining showed that the integrity of intestinal organoids in the 5% concentration serum group was better than that in the 10% and 20% groups. The 5% concentration serum group showed the highest number of organoids, the highest germination rate and the lowest pathological score among experimental groups, while the 20% group exhibited severe morphological destruction and almost no germination was observed, and the pathological score was the highest among all groups(t=3.668, 4.334,5.309,P<0.05). The results of Alican blue staining showed that the secretion function of intestinal organoids in serum culture of aGVHD in the 20% group was weaker than that of the 5% group and 10% of the organoids, and there was almost no goblet cells, and mucus was stainned in the 20% aGVHD serum group. The immunohistochemical results showed that the number of Lgr5+ cells of intestinal organoids in the 5% group was more than that of the intestinal organoids in the 10% aGVHD serum group and 20% aGVHD serum group. Almost no Clu+ cells were observed in the 5% group. The Lgr5+ cells in the 20% group were seriously injuried and can not be observed. The proportion of Clu+ cells in the 20% group significantly increased.@*CONCLUSION@#The concentration of aGVHD serum in the culture system can affect the number and secretion function of intestinal organoids as well as the number of intestinal stem cells in organoids. The higher the serum concentration, the greater the risk of organoid injury, which reveal the characteristics of the formation and functional change of aGVHD intestinal organoids, and provide a novel tool for the study of intestinal injury in aGVHD.

Erythropoietin promotes myocardial infarction repair in mice by improving the function of Sca-1+ stem cells / 生理学报

Myocardial infarction (MI) is one of the leading causes of death in the world. With the improvement of clinical therapy, the mortality of acute MI has been significantly reduced. However, as for the long-term impact of MI on cardiac remodeling and cardiac function, there is no effective prevention and treatment measures. Erythropoietin (EPO), a glycoprotein cytokine essential to hematopoiesis, has anti-apoptotic and pro-angiogenetic effects. Studies have shown that EPO plays a protective role in cardiomyocytes in cardiovascular diseases, such as cardiac ischemia injury and heart failure. EPO has been demonstrated to protect ischemic myocardium and improve MI repair by promoting the activation of cardiac progenitor cells (CPCs). This study aimed to investigate whether EPO can promote MI repair by enhancing the activity of stem cell antigen 1 positive stem cells (Sca-1+ SCs). Darbepoetin alpha (a long-acting EPO analog, EPOanlg) was injected into the border zone of MI in adult mice. Infarct size, cardiac remodeling and performance, cardiomyocyte apoptosis and microvessel density were measured. Lin- Sca-1+ SCs were isolated from neonatal and adult mouse hearts by magnetic sorting technology, and were used to identify the colony forming ability and the effect of EPO, respectively. The results showed that, compared to MI alone, EPOanlg reduced the infarct percentage, cardiomyocyte apoptosis ratio and left ventricular (LV) chamber dilatation, improved cardiac performance, and increased the numbers of coronary microvessels in vivo. In vitro, EPO increased the proliferation, migration and clone formation of Lin- Sca-1+ SCs likely via the EPO receptor and downstream STAT-5/p38 MAPK signaling pathways. These results suggest that EPO participates in the repair process of MI by activating Sca-1+ SCs.

Effects and mechanism of p53 gene deletion on energy metabolism during the pluripotent transformation of spermatogonial stem cells / 生理学报

Previous studies have shown that long-term spermatogonial stem cells (SSCs) have the potential to spontaneously transform into pluripotent stem cells, which is speculated to be related to the tumorigenesis of testicular germ cells, especially when p53 is deficient in SSCs which shows a significant increase in the spontaneous transformation efficiency. Energy metabolism has been proved to be strongly associated with the maintenance and acquisition of pluripotency. Recently, we compared the difference in chromatin accessibility and gene expression profiles between wild-type (p53+/+) and p53 deficient (p53-/-) mouse SSCs using the Assay for Targeting Accessible-Chromatin with high-throughput sequencing (ATAC-seq) and transcriptome sequencing (RNA-seq) techniques, and revealed that SMAD3 is a key transcription factor in the transformation of SSCs into pluripotent cells. In addition, we also observed significant changes in the expression levels of many genes related to energy metabolism after p53 deletion. To further reveal the role of p53 in the regulation of pluripotency and energy metabolism, this paper explored the effects and mechanism of p53 deletion on energy metabolism during the pluripotent transformation of SSCs. The results of ATAC-seq and RNA-seq from p53+/+ and p53-/- SSCs revealed that gene chromatin accessibility related to positive regulation of glycolysis and electron transfer and ATP synthesis was increased, and the transcription levels of genes encoding key glycolytic enzymes and regulating electron transport-related enzymes were markedly increased. Furthermore, transcription factors SMAD3 and SMAD4 promoted glycolysis and energy homeostasis by binding to the chromatin of the Prkag2 gene which encodes the AMPK subunit. These results suggest that p53 deficiency activates the key enzyme genes of glycolysis in SSCs and enhances the chromatin accessibility of genes associated with glycolysis activation to improve glycolysis activity and promote transformation to pluripotency. Moreover, SMAD3/SMAD4-mediated transcription of the Prkag2 gene ensures the energy demand of cells in the process of pluripotency transformation and maintains cell energy homeostasis by promoting AMPK activity. These results shed light on the importance of the crosstalk between energy metabolism and stem cell pluripotency transformation, which might be helpful for clinical research of gonadal tumors.

Mechanism of Notch3 signaling pathway regulating the differentiation of aortic dissection vascular stem cells into smooth muscle cells / 中华危重病急救医学

OBJECTIVE@#To explore whether the differentiation of vascular stem cells (VSC) into smooth muscle cells (SMC) in aortic dissection (AD) is dysregulated, and to verify the role of Notch3 pathway in this process.@*METHODS@#Aortic tissues were obtained from AD patients undergoing aortic vascular replacement and heart transplant donors at Department of Cardiovascular Surgery, Guangdong Provincial People's Hospital Affiliated to Southern Medical University. VSC were isolated by enzymatic digestion and c-kit immunomagnetic beads. The cells were divided into normal donor-derived VSC group (Ctrl-VSC group) and AD-derived VSC group (AD-VSC group). The presence of VSC in the aortic adventitia was detected by immunohistochemical staining, and VSC was identified by stem cell function identification kit. The differentiation model of VSC into SMC established in vitro was induced by transforming growth factor-β1 (10 μg/L) for 7 days. They were divided into normal donor VSC-SMC group (Ctrl-VSC-SMC group), AD VSC-SMC group (AD-VSC-SMC group) and AD VSC-SMC+Notch3 inhibitor DAPT group (AD-VSC-SMC+DAPT group,DAPT 20 μmol/L was added during differentiation induction). The expression of contractile marker Calponin 1 (CNN1) in SMC derived from aortic media and VSC were detected by immunofluorescence staining. The protein expressions of contractile markers α-smooth muscle actin (α-SMA), CNN1 as well as Notch3 intracellular domain (NICD3) in SMC derived from aortic media and VSC were detected by Western blotting.@*RESULTS@#Immunohistochemical staining showed there was a population of c-kit-positive VSC in the adventitia of aortic vessels, and VSC from both normal donors and AD patients had the ability to differentiate into adipocytes and chondrocytes. Compared with normal donor vascular tissue, the expressions of SMC markers α-SMA and CNN1 of tunica media contraction in AD were down-regulated (α-SMA/β-actin: 0.40±0.12 vs. 1.00±0.11, CNN1/β-actin: 0.78±0.07 vs. 1.00±0.14, both P < 0.05), while the protein expression of NICD3 was up-regulated (NICD3/GAPDH: 2.22±0.57 vs. 1.00±0.15, P < 0.05). Compared with Ctrl-VSC-SMC group, the expressions of contractile SMC markers α-SMA and CNN1 were down-regulated in AD-VSC-SMC group (α-SMA/β-actin: 0.35±0.13 vs. 1.00±0.20, CNN1/β-actin: 0.78±0.06 vs. 1.00±0.07, both P < 0.05), the protein expression of NICD3 was up-regulated (NICD3/GAPDH: 22.32±1.22 vs. 1.00±0.06, P < 0.01). Compared with AD-VSC-SMC group, the expressions of contractile SMC markers α-SMA, CNN1 were up-regulated in AD-VSC-SMC+DAPT group (α-SMA/β-actin: 1.70±0.07 vs. 1.00±0.15, CNN1/β-actin: 1.62±0.03 vs. 1.00±0.02, both P < 0.05).@*CONCLUSIONS@#Dysregulation of VSC differentiation into SMC occurs in AD, while inhibition of Notch3 pathway activation can restore the expression of contractile proteins in VSC-derived SMC in AD.

Liver cell therapies: cellular sources and grafting strategies / 医学前沿

The liver has a complex cellular composition and a remarkable regenerative capacity. The primary cell types in the liver are two parenchymal cell populations, hepatocytes and cholangiocytes, that perform most of the functions of the liver and that are helped through interactions with non-parenchymal cell types comprising stellate cells, endothelia and various hemopoietic cell populations. The regulation of the cells in the liver is mediated by an insoluble complex of proteins and carbohydrates, the extracellular matrix, working synergistically with soluble paracrine and systemic signals. In recent years, with the rapid development of genetic sequencing technologies, research on the liver's cellular composition and its regulatory mechanisms during various conditions has been extensively explored. Meanwhile breakthroughs in strategies for cell transplantation are enabling a future in which there can be a rescue of patients with end-stage liver diseases, offering potential solutions to the chronic shortage of livers and alternatives to liver transplantation. This review will focus on the cellular mechanisms of liver homeostasis and how to select ideal sources of cells to be transplanted to achieve liver regeneration and repair. Recent advances are summarized for promoting the treatment of end-stage liver diseases by forms of cell transplantation that now include grafting strategies.

Human menstrual blood-derived stem cells alleviate autoimmune hepatitis via JNK/MAPK signaling pathway in vivo and in vitro / 医学前沿

Autoimmune hepatitis (AIH) is a severe globally distributed liver disease that could occur at any age. Human menstrual blood-derived stem cells (MenSCs) have shown therapeutic effect in acute lung injury and liver failure. However, their role in the curative effect of AIH remains unclear. Here, a classic AIH mouse model was constructed through intravenous injection with concanavalin A (Con A). MenSCs were intravenously injected while Con A injection in the treatment groups. The results showed that the mortality by Con A injection was significantly decreased by MenSCs treatment and liver function tests and histological analysis were also ameliorated. The results of phosphoproteomic analysis and RNA-seq revealed that MenSCs improved AIH, mainly by apoptosis and c-Jun N-terminal kinase/mitogen-activated protein signaling pathways. Apoptosis analysis demonstrated that the protein expression of cleaved caspase 3 was increased by Con A injection and reduced by MenSCs transplantation, consistent with the TUNEL staining results. An AML12 co-culture system and JNK inhibitor (SP600125) were used to verify the JNK/MAPK and apoptosis signaling pathways. These findings suggested that MenSCs could be a promising strategy for AIH.

Spatially resolved expression landscape and gene-regulatory network of human gastric corpus epithelium

Molecular knowledge of human gastric corpus epithelium remains incomplete. Here, by integrated analyses using single-cell RNA sequencing (scRNA-seq), spatial transcriptomics, and single-cell assay for transposase accessible chromatin sequencing (scATAC-seq) techniques, we uncovered the spatially resolved expression landscape and gene-regulatory network of human gastric corpus epithelium. Specifically, we identified a stem/progenitor cell population in the isthmus of human gastric corpus, where EGF and WNT signaling pathways were activated. Meanwhile, LGR4, but not LGR5, was responsible for the activation of WNT signaling pathway. Importantly, FABP5 and NME1 were identified and validated as crucial for both normal gastric stem/progenitor cells and gastric cancer cells. Finally, we explored the epigenetic regulation of critical genes for gastric corpus epithelium at chromatin state level, and identified several important cell-type-specific transcription factors. In summary, our work provides novel insights to systematically understand the cellular diversity and homeostasis of human gastric corpus epithelium in vivo.

Autologous mesenchymal stem cells offer a new paradigm for salivary gland regeneration / 国际口腔科学杂志·英文版

Salivary gland (SG) dysfunction, due to radiotherapy, disease, or aging, is a clinical manifestation that has the potential to cause severe oral and/or systemic diseases and compromise quality of life. Currently, the standard-of-care for this condition remains palliative. A variety of approaches have been employed to restore saliva production, but they have largely failed due to damage to both secretory cells and the extracellular matrix (niche). Transplantation of allogeneic cells from healthy donors has been suggested as a potential solution, but no definitive population of SG stem cells, capable of regenerating the gland, has been identified. Alternatively, mesenchymal stem cells (MSCs) are abundant, well characterized, and during SG development/homeostasis engage in signaling crosstalk with the SG epithelium. Further, the trans-differentiation potential of these cells and their ability to regenerate SG tissues have been demonstrated. However, recent findings suggest that the "immuno-privileged" status of allogeneic adult MSCs may not reflect their status post-transplantation. In contrast, autologous MSCs can be recovered from healthy tissues and do not present a challenge to the recipient's immune system. With recent advances in our ability to expand MSCs in vitro on tissue-specific matrices, autologous MSCs may offer a new therapeutic paradigm for restoration of SG function.

Overview of the main biological mechanisms linked to changes in periodontal ligament stem cells and the inflammatory microenvironment / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Periodontitis is a complex chronic inflammatory disease. The invasion of pathogens induces the inflammatory microenvironment in periodontitis. Cell behavior changes in response to changes in the microenvironment, which in turn alters the local inflammatory microenvironment of the periodontium through factors secreted by cells. It has been confirmed that periodontal ligament stem cells (PDLSCs) are vital in the development of periodontal disease. Moreover, PDLSCs are the most effective cell type to be used for periodontium regeneration. This review focuses on changes in PDLSCs, their basic biological behavior, osteogenic differentiation, and drug effects caused by the inflammatory microenvironment, to provide a better understanding of the influence of these factors on periodontal tissue homeostasis. In addition, we discuss the underlying mechanism in detail behind the reciprocal responses of PDLSCs that affect the microenvironment.

FOXP4 promotes proliferation of human spermatogonial stem cells / 亚洲男科学杂志(英文版)

Continuous self-renewal and differentiation of spermatogonial stem cells (SSCs) is vital for maintenance of adult spermatogenesis. Although several spermatogonial stem cell regulators have been extensively investigated in rodents, regulatory mechanisms of human SSC self-renewal and differentiation have not been fully established. We analyzed single-cell sequencing data from the human testis and found that forkhead box P4 (FOXP4) expression gradually increased with development of SSCs. Further analysis of its expression patterns in human testicular tissues revealed that FOXP4 specifically marks a subset of spermatogonia with stem cell potential. Conditional inactivation of FOXP4 in human SSC lines suppressed SSC proliferation and significantly activated apoptosis. FOXP4 expressions were markedly suppressed in tissues with dysregulated spermatogenesis. These findings imply that FOXP4 is involved in human SSC proliferation, which will help elucidate on the mechanisms controlling the fate decisions in human SSCs.