BACKGROUND: The effects of human umbilical cord-derived mesenchymal stem cell (UC-MSC) treatment on coronavirus disease 2019 (COVID-19) patients have been preliminarily characterized. However, real-world data on the safety and efficacy of intravenous transfusions of MSCs in hospitalized COVID-19 patients at the convalescent stage remain to be reported. METHODS: This was a single-arm, multicenter, real-word study in which a contemporaneous external control was included as the control group. Besides, severe and critical COVID-19 patients were considered together as the severe group, given the small number of critical patients. For a total of 110 patients, 21 moderate patients and 31 severe patients were enrolled in the MSC treatment group, while 26 moderate patients and 32 severe patients were enrolled in the control group. All patients received standard treatment. The MSC treatment patients additionally received intravenous infusions of MSCs at a dose of 4 × 10 7 cells on days 0, 3, and 6, respectively. The clinical outcomes, including adverse events (AEs), lung lesion proportion on chest computed tomography, pulmonary function, 6-min walking distance (6-MWD), clinical symptoms, and laboratory parameters, were measured on days 28, 90, 180, 270, and 360 during the follow-up visits. RESULTS: In patients with moderate COVID-19, MSC treatment improved pulmonary function parameters, including forced expiratory volume in the first second (FEV1) and maximum forced vital capacity (VCmax) on days 28 (FEV1, 2.75 [2.35, 3.23] vs . 2.11 [1.96, 2.35], P  = 0.008; VCmax, 2.92 [2.55, 3.60] vs . 2.47 [2.18, 2.68], P  = 0.041), 90 (FEV1, 2.93 [2.63, 3.27] vs . 2.38 [2.24, 2.63], P  = 0.017; VCmax, 3.52 [3.02, 3.80] vs . 2.59 [2.45, 3.15], P  = 0.017), and 360 (FEV1, 2.91 [2.75, 3.18] vs . 2.30 [2.16, 2.70], P  = 0.019; VCmax,3.61 [3.35, 3.97] vs . 2.69 [2.56, 3.23], P  = 0.036) compared with the controls. In addition, in severe patients, MSC treatment notably reduced the proportion of ground-glass lesions in the whole lung volume on day 90 ( P  = 0.045) compared with the controls. No difference in the incidence of AEs was observed between the two groups. Similarly, no significant differences were found in the 6-MWD, D-dimer levels, or interleukin-6 concentrations between the MSC and control groups. CONCLUSIONS: Our results demonstrate the safety and potential of MSC treatment for improved lung lesions and pulmonary function in convalescent COVID-19 patients. However, comprehensive and long-term studies are required to confirm the efficacy of MSC treatment. TRIAL REGISTRATION Chinese Clinical Trial Registry, ChiCTR2000031430.
Humans ; COVID-19/therapy* ; Female ; Male ; Mesenchymal Stem Cell Transplantation/adverse effects* ; Middle Aged ; Adult ; Umbilical Cord/cytology* ; Mesenchymal Stem Cells/cytology* ; SARS-CoV-2 ; Aged ; Treatment Outcome

Infertility has become one of the most serious diseases worldwide, and 50% of this disease can be attributed to male-related factors. Spermatogenesis, by definition, is a complex process by which spermatogonial stem cells (SSCs) self-renew to maintain stem cell population within the testes and differentiate into mature spermatids. It is of great significance to uncover gene regulation and signaling pathways that are involved in the fate determinations of SSCs with aims to better understand molecular mechanisms underlying human spermatogenesis and identify novel targets for gene therapy of male infertility. Significant achievement has recently been made in demonstrating the signaling molecules and pathways mediating the fate decisions of mammalian SSCs. In this review, we address key gene regulation and crucial signaling transduction pathways in controlling the self-renewal, differentiation, and apoptosis of SSCs, and we illustrate the networks of genes and signaling pathways in SSC fate determinations. We also highlight perspectives and future directions in SSC regulation by genes and their signaling pathways. This review could provide novel insights into the genetic regulation of normal and abnormal spermatogenesis and offer molecular targets to develop new approaches for gene therapy of male infertility.
Humans ; Male ; Signal Transduction/physiology* ; Apoptosis/physiology* ; Spermatogenesis/physiology* ; Cell Differentiation ; Adult Germline Stem Cells/physiology* ; Spermatogonia/cytology* ; Gene Expression Regulation ; Animals ; Infertility, Male/genetics* ; Cell Self Renewal/genetics*

Icariin is a pure compound derived from Epimedium brevicornu Maxim, and it helps the regulation of male reproduction. Nevertheless, the role and underlying mechanisms of Icariin in mediating male germ cell development remain to be clarified. Here, we have demonstrated that Icariin promoted proliferation and DNA synthesis of mouse spermatogonial stem cells (SSCs). Furthermore, surface plasmon resonance iron (SPRi) and molecular docking (MOE) assays revealed that phosphodiesterase 5A (PDE5A) was an important target of Icariin in mouse SSCs. Mechanically, Icariin decreased the expression level of PDE5A. Interestingly, hydrogen peroxides (H 2 O 2 ) enhanced the expression level of phosphorylation H2A.X (p-H2A.X), whereas Icariin diminished the expression level of p-H2A.X and DNA damage caused by H 2 O 2 in mouse SSCs. Finally, our in vivo animal study indicated that Icariin protected male reproduction. Collectively, these results implicate that Icariin targets PDE5A to regulate mouse SSC viability and DNA damage and improves male reproductive capacity. This study thus sheds new insights into molecular mechanisms underlying the fate decisions of mammalian SSCs and offers a scientific basis for the clinical application of Icariin in male reproduction.
Male ; Animals ; Flavonoids/pharmacology* ; Mice ; Cyclic Nucleotide Phosphodiesterases, Type 5/drug effects* ; DNA Damage/drug effects* ; Cell Survival/drug effects* ; Cell Proliferation/drug effects* ; Spermatogonia/drug effects* ; Reproduction/drug effects* ; Adult Germline Stem Cells/metabolism* ; DNA Replication/drug effects*

OBJECTIVE: To analyze the Epstein-Barr virus (EBV) load in different lymphocyte subsets, as well as clinical characteristics and outcomes in patients with hematologic malignancies experiencing EBV reactivation. METHODS: Peripheral blood samples from patients were collected. B, T, and NK cells were isolated sorting with magnetic beads by flow cytometry. The EBV load in each subset was quantitated by real-time quantitative polymerase chain reaction (RT-qPCR). Clinical data were colleted from electronic medical records. Survival status was followed up through outpatient visits and telephone calls. Statistical analyses were performed using SPSS 25.0. RESULTS: A total of 39 patients with hematologic malignancies were included, among whom 35 patients had undergone allogeneic hematopoietic stem cell transplantation (allo-HSCT). The median time to EBV reactivation was 4.8 months (range: 1.7-57.1 months) after allo-HSCT. EBV was detected in B, T, and NK cells in 20 patients, in B and T cells in 11 patients, and only in B cells in 4 patients. In the 35 patients, the median EBV load in B cells was 2.19×10⁴ copies/ml, significantly higher than that in T cells (4.00×10³ copies/ml, P <0.01) and NK cells (2.85×10² copies/ml, P <0.01). Rituximab (RTX) was administered for 32 patients, resulting in EBV negativity in 32 patients with a median time of 8 days (range: 2-39 days). Post-treatment analysis of 13 patients showed EBV were all negative in B, T, and NK cells. In the four non-transplant patients, the median time to EBV reactivation was 35 days (range: 1-328 days) after diagnosis of the primary disease. EBV was detected in one or two subsets of B, T, or NK cells, but not simultaneously in all three subsets. These patients received a combination chemotherapy targeting at the primary disease, with 3 patients achieving EBV negativity, and the median time to be negative was 40 days (range: 13-75 days). CONCLUSION In hematologic malignancy patients after allo-HSCT, EBV reactivation commonly involves B, T, and NK cells, with a significantly higher viral load in B cells compared to T and NK cells. Rituximab is effective for EBV clearance. In non-transplant patients, EBV reactivation is restricted to one or two lymphocyte subsets, and clearance is slower, highlighting the need for prompt anti-tumor therapy.
Humans ; Hematologic Neoplasms/virology* ; Herpesvirus 4, Human/physiology* ; Epstein-Barr Virus Infections ; Hematopoietic Stem Cell Transplantation ; Virus Activation ; Lymphocyte Subsets/virology* ; Flow Cytometry ; Killer Cells, Natural/virology* ; Male ; Female ; B-Lymphocytes/virology* ; Viral Load ; Adult ; T-Lymphocytes/virology* ; Middle Aged

Lumbar disc (LD) herniation and aging are prevalent conditions that can result in substantial morbidity. This study aimed to clarify the mechanisms connecting the LD aging and herniation, particularly focusing on cellular senescence and molecular alterations in the nucleus pulposus (NP). We performed a detailed analysis of NP samples from a diverse cohort, including individuals of varying ages and those with diagnosed LD herniation. Our methodology combined histological assessments with single-nucleus RNA sequencing to identify phenotypic and molecular changes related to NP aging and herniation. We discovered that cellular senescence and a decrease in nucleus pulposus progenitor cells (NPPCs) are central to both processes. Additionally, we found an age-related increase in NFAT1 expression that promotes NPPC senescence and contributes to both aging and herniation of LD. This research offers fresh insights into LD aging and its associated pathologies, potentially guiding the development of new therapeutic strategies to target the root causes of LD herniation and aging.
Intervertebral Disc Displacement/metabolism* ; Humans ; Aging/pathology* ; Nucleus Pulposus/pathology* ; Male ; Female ; Transcriptome ; Middle Aged ; Lumbar Vertebrae/pathology* ; Adult ; Cellular Senescence ; Stem Cells/pathology* ; Aged ; Intervertebral Disc Degeneration/metabolism*

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.
Adult ; Humans ; Male ; Cell Differentiation ; Cell Proliferation ; Forkhead Transcription Factors/metabolism* ; Spermatogenesis/genetics* ; Spermatogonia/metabolism* ; Stem Cells/metabolism* ; Testis/metabolism*

OBJECTIVE: To explore the clinical characteristics, treatment, and prognosis of patients with blastic plasmacytoid dendritic cell neoplasm(BPDCN). METHODS: Clinical data of 5 patients diagnosed with BPDCN in Wuhan First Hospital and Wuhan Tongji Hospital from June 2016 to November 2021 were retrospectively analyzed. RESULTS: Among the 5 patients, 3 were male and 2 were female, with a median age of 28(10-52) years old. Four patients showed obvious skin damage at the initial diagnosis; the other one showed clinical manifestations of acute leukemia rather than obvious skin damage at the initial diagnosis, but infiltrated skin when the disease relapsed after treatment. Other infiltration sites of lesions included bone marrow (2/5), peripheral blood (2/5), lymph nodes (3/5), liver and spleen (2/5). All patients had no clinical manifestation of central nervous system infiltration. Tumor cell specific immune markers CD4, CD56, CD123 were all positive, and the median Ki-67 index was 70%. TET2, ASXL1 and NRAS gene mutations were found respectively in 3 patients by next-generation sequencing technique (NGS). ALL-like, AML-like and invasive NK/T cell lymphoma-like first-line induction chemotherapy regimens were used for the patients. One patient died of severe complications during the early stage of chemotherapy, 3 patients were evaluated as CR, and 1 patient was evaluated as PR. 2 patients were recurred and progressed after induction of chemotherapy, and one of them was evaluated as CR after re-treatment. One patient received autologous hematopoietic stem cell transplantation (auto-HSCT) and got long-term survival (OS 87 months). 3 patients received allogeneic hematopoietic stem cell transplantation (allo-HSCT), of which one died of transplantation related complications, and 2 cases survived. The median follow-up time of 4 patients with evaluable efficacy was 28.5(9-84) months, the median OS time was 31.5(10-87) months. CONCLUSION BPDCN is a highly heterogeneous malignant tumor with a poor prognosis. HSCT, especially allo-HSCT can significantly improve the prognosis of BPDCN patients.
Humans ; Male ; Female ; Adult ; Middle Aged ; Retrospective Studies ; Leukemia/pathology* ; Hematopoietic Stem Cell Transplantation ; Prognosis ; Myeloproliferative Disorders ; Skin Neoplasms/pathology* ; Acute Disease ; Dendritic Cells

OBJECTIVE: To investigate the efficacy and safety of chemotherapy combined with venetoclax followed by allogeneic hematopoietic stem cell transplantation (allo-HSCT) for the treatment of blastic plasmacytoid dendritic cell neoplasm (BPDCN). METHODS: The clinical data of 3 patients with BPDCN undergoing allo-HSCT in Department of Hematology, Wuhan First Hospital from July 2017 to November 2021 were collected and retrospectively analyzed. RESULTS: Among the 3 patients, there were 1 male and 2 females, aged 27-52 years old. Skin lesions were observed during initial diagnosis, and it could also be characterized by acute leukemia. Characteristic molecular markers of tumor cells, such as CD4, CD56, CD123, and CD303 were positive. In addition, the expression detection of Bcl-2 in 3 patients were positive. Chemotherapy combined with venetoclax in the initial induction of chemotherapy (1 case) or disease recurrence and progress (2 cases) was performed. There were 2 cases evaluated as complete remission (CR) and 1 case as partial remission (PR) before allo-HSCT. The patients all received a nonmyeloablative conditioning without total body irradiation (TBI). The prevention programme of graft-versus-host disease (GVHD) was antithymocyte globulin + mycophenolate mofetil + cyclosporin A/FK506 ± methotrexate. The number of mononuclear cell (MNC) count was (16.73-18.35)×10⁸/kg, and CD34⁺ cell count was (3.57-4.65)×10⁶/kg. The 3 patients were evaluated as CR after allo-HSCT (+21 to +28 d), the donor-recipient chimerism rate was 100%, and Ⅲ-Ⅳ GVHD was not observed. One patient died at +50 d after transplantation, two patients were followed up for 28 months and 15 months, respectively, and achieved disease-free survival (DFS). CONCLUSIONS BPDCN is a highly aggressive malignant tumor with poor prognosis. Chemotherapy combined with venetoclax followed by allo-HSCT may lead to long-term DFS or even cure. Post-transplant maintenance is still unclear.
Female ; Humans ; Male ; Adult ; Middle Aged ; Retrospective Studies ; Hematopoietic Stem Cell Transplantation/adverse effects* ; Acute Disease ; Graft vs Host Disease/prevention & control* ; Myeloproliferative Disorders ; Leukemia, Myeloid, Acute/pathology* ; Dendritic Cells

OBJECTIVE: To analyze the prognostic factors of haplo-HSCT combined with MSC in the treatment of SAA. METHODS: 127 SAA patients who had undergone haplo-HSCT with co-infusion of MSC in our center from January 2014 to August 2019 were analyzed retrospectively. Median age was 11 (1-37) years, and median follow-up time was 39.8 (1-74) months. RESULTS: The median time for neutrophil and platelet engraftment was 14 d and 18 d respectively. The cumulative incidences of grade III-IV aGVHD was 4.4%±1.9% at day +100. The 2-year cumulative incidence of extensive cGVHD was 8.3%± 2.7%. The estimated 3-year OS was 86.1%±3.1%. Univariate analysis showed that high-dose CD34⁺ cells (>6.69×106/kg) could promote the engraftment of neutrophil (97.9%±0.05% vs 88.6%±0.13% at day +21, P=0.0006) and platelet (81.2%±0.33% vs 70.8%±0.26% at day +28, P=0.002) and did not increase the incidence of aGVHD (10.4%±0.1% vs 18.9%±0.1% at day +100, P=0.18). More nucleated cells (>12.78×10⁸/kg) caused a lower incidence of grade II-IV aGVHD (8.6%±0.13% vs 21.7%±0.25% at day+100, P=0.04) and a higher incidence of 3-year OS (91.3%±3.2% vs 78.1%±6.5%, P=0.03) than less nucleated cells (≤12.78×10⁸/kg). Younger patients (age≤12 y) had faster neutrophil engraftment (94.9%±0.06% vs 87.5%±0.24% at day+21, P=0.02), higher 3-year OS (93.6%±2.8% vs 75.9%±6.4%, P=0.006) and higher 3-year FFS (93.6%±2.8% vs 68.3%±7.1%, P=0.000) than older patients (age>12 y). The shorter the time from diagnosis to HSCT (≤29.5 months), the higher the 3-year FFS of patients (88.8%±3.5% vs 74.2%±7.2%, P=0.028). Male patients with female donors had higher cumulative incidence of extensive cGVHD than others (20.0%±0.8% vs 4.6%±0.1%, P=0.01). CONCLUSION In the haplo-HSCT of SAA, the prognosis of children patients is better than that of adults patients. More CD34⁺ cells and nucleated cells can promote engraftment, reduce the incidence of aGVHD and improve OS. HSCT should be performed as early as possible, and the occurrence of cGVHD should be reduced in male patients by avoiding female donors.
Adult ; Anemia, Aplastic/therapy* ; Child ; Female ; Graft vs Host Disease ; Hematopoietic Stem Cell Transplantation/adverse effects* ; Humans ; Male ; Mesenchymal Stem Cells ; Retrospective Studies ; Transplantation Conditioning/adverse effects* ; Treatment Outcome

Objective: To investigate the expression and function of collagen type ⅩⅦ α1 (COL17α1) in aging mouse skin and its effect on the stemness and proliferation of human epidermal stem cells (ESCs), and to explore the mechanism of related microRNA (miR) in intervening the expression of COL17α1 of human ESC. Methods: The method of experimental research was used. Twelve 2-month-old (young) and twelve 24-month-old (aged) male C57BL/6J mice were selected, and full-thickness skin samples from their upper back were taken for follow-up detection. After hematoxylin-eosin staining of the full-thickness skin samples of young mice and aged mice, the structure of the epidermis was observed and the thickness of the epidermis was measured; the morphology of epidermal basement membrane and hemidesmosomes were observed by transmission electron microscopy, and the hemidesmosomes were counted; the mRNA and protein expressions of COL17α1 were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blotting respectively, and the protein expression and distribution of COL17α1 was observed and detected by immunofluorescence method. The fresh foreskin tissue discarded after surgery was obtained from 3 healthy men aged 20-30 years who underwent circumcision at the Fourth Medical Center of PLA General Hospital, ESCs were extracted and well-grown cells were wsed for follow-up experiments. According to the random number table (the same grouping method below), ESCs were divided into blank control group, transfection reagent control group, empty vector plasmid group, and COL17α1 knockdown plasmid group with corresponding treatment. After 48 hours of culture, the mRNA expression of COL17α1 was detected by real-time fluorescent quantitative RT-PCR, the protein expressions of COL17α1 and cytokeratin 14 (CK14) were detected by Western blotting, and the cell proliferation level was detected by cell counting kit 8. miRs that might act on the 3' non-coding region of COL17α1 mRNA were screened through DIANA, miRTarBase, miRNAMap, TargetScan, and microRNA databases. The ESCs were divided into negative control group transfected with miR mimic negative control and each miR mimic group transfected with each of the previously screened miR mimics. Forty-eight hours after transfection, the protein expression of COL17α1 was detected by Western blotting. Based on the sequencing data set GSE114006 in Gene Expression Omnibus (GEO), the GEO2R tool was used to statistically analyze the expression of the previously screened miRs that could cause the reduction of COL17α1 protein expression in the skin of 30 young (18-25 years old) and 30 elderly (>70 years old) human skins. The full-thickness skin samples of young mice and aged mice were taken, and the expressions of increased miRs in the aforementioned aged human skin were detected by real-time fluorescent quantitative RT-PCR. Two batches of human ESCs were taken, the first batch was divided into COL17α1 wild type+miR-203b-3p negative control group and COL17α1 wild type+miR-203b-3p mimic group, and the second batch was divided into COL17α1 mutant+miR-203b-3p negative control group and COL17α1 mutant+miR-203b-3p mimic group. Each group of ESC was transfected with corresponding sequences respectively. Forty-eight hours later, the luciferase reporter gene detection kit was used to detect the gene expression level of COL17α1. The number of samples in the tissue experiment was 6, and the number of samples in the cell experiment was 3. Data were statistically analyzed with independent sample t test, one-way analysis of variance, least significant difference test or Dunnett's test, Mann-Whitney U test or Kruskal-Wallis H test. Results: Compared with those of young mice, the boundary between the epidermis and the dermis of the aged mice skin was blurred and the cell layers were less, and the thickness of epidermis was significantly thinner (Z=-2.88, P<0.01); the morphology of basement membrane was discontinuous, with less unevenly distributed hemidesmosomes at the epidermis-dermis junction, and the number of hemidesmosomes was significantly reduced (Z=-2.91, P<0.01); the mRNA and protein expression levels of COL17α1 in the skin of aged mice were significantly decreased (with t values of 10.61 and 6.85, respectively, P<0.01). Compared with those of young mice, the protein expression of COL17α1 in the basal layer of epidermis and the bulb of hair follicle in the skin of aged mice was significantly decreased (Z=-2.24, P<0.05). After 48 hours of culture, the protein expression levels of COL17α1 in ESCs of blank control group, transfection reagent control group, empty vector plasmid group, and COL17α1 knockdown plasmid group were 1.00±0.27, 1.12±0.21, 1.13±0.23, and 0.42±0.18, respectively. Compared with those of blank control group, the mRNA and protein expression levels of COL17α1, the protein expression level of CK14, and the proliferation level of ESCs in transfection reagent control group and empty vector plasmid group did not change significantly (P>0.05), while these indexes in COL17α1 knockdown plasmid group were significantly decreased (P<0.05 or P<0.01). miR-203a-3p, miR-203b-3p, miR-512-5p, miR-124-3p, miR-28-5p, miR-590-3p, and miR-329-5p might bind to the 3' non-coding region of COL17α1 mRNA. Forty-eight hours after transfection, compared with 1.000±0.224 in negative control group, the protein expression level of COL17α1 in ESCs of miR-329-5p mimic group, miR-203b-3p mimic group, and miR-203a-3p mimic group decreased significantly (0.516±0.188, 0.170±0.025, and 0.235±0.025, with t values of 3.17, 5.43, and 5.07, respectively, P<0.05 or P<0.01). Only the expression level of miR-203b-3p in the skin of the elderly was significantly higher than that of the young (t=3.27, P<0.01). The expression level of miR-203b-3p in the skin of aged mice was significantly higher than that of young mice (Z=-2.88, P<0.01). Forty-eight hours after transfection, the gene expression level of COL17α1 in ESCs of COL17α1 wild type+miR-203b-3p mimic group was significantly lower than that of COL17α1 wild type+miR-203b-3p negative control group (t=7.66, P<0.01). The gene expression level of COL17α1 in ESCs of COL17α1 mutant+miR-203b-3p mimic group was similar to that of COL17α1 mutant+miR-203b-3p negative control group (P>0.05). Conclusions: The mRNA and protein expression levels of COL17α1 decrease with age increasing in mice, which may lead to the detachment of mouse ESC from the epidermal basement membrane. Decreased expression of COL17α1 can inhibit the expression of CK14 and ESC proliferation, which may be responsible for the thinning of the epidermis and slower wound healing in aged human skin. The increased expression of miR-203b-3p in aged mouse skin can target and bind to the 3' non-coding region of COL17α1 mRNA, hindering the post-transcriptional translation process, thus resulting in decreased COL17α1 protein expression.
Adolescent ; Adult ; Aged ; Animals ; Autoantigens ; Humans ; Keratin-14 ; Male ; Mice ; Mice, Inbred C57BL ; MicroRNAs/genetics* ; Non-Fibrillar Collagens/pharmacology* ; Polyesters ; RNA, Messenger ; Skin Aging ; Stem Cells ; Young Adult