衰老导致卵巢功能低下研究进展

由于社会角色的转变,女性生育延迟现象明显。女性卵巢功能一般从35岁时开始下降,主要表现为卵泡数量减少和卵母细胞质量下降。目前临床上对于卵巢功能低下的诊断主要依据血清卵泡刺激素(follicle stimulating hormone,FSH)、血清抗苗勒氏管激素(anti-Müllerian hormone,AMH)、窦卵泡计数、年龄、月经和抑制素B等指标。目前研究发现,伴随年龄的增加,女性卵巢内细胞会出现线粒体功能失调、染色质短缩、DNA修复减少、表观遗传学改变和代谢失序。本文在简要介绍卵巢功能低下临床诊断的基础上,对衰老导致卵巢功能低下的相关因素进行了总结,并深入探讨了其发生的分子机制及潜在的干预靶点,以期为有效改善高龄女性的卵巢功能提供思路。

Effect of autologous bone marrow stem cells-scaffold transplantation on the ongoing pregnancy rate in intrauterine adhesion women:a randomized,controlled trial

Intrauterine adhesion is a major cause of female reproductive disorders.Although we and others uncontrolled pilot studies showed that treatment with autologous bone marrow stem cells made a few patients with severe intrauterine adhesion obtain live birth,no large sample randomized controlled studies on this therapeutic strategy in such patients have been reported so far.To verify if the therapy of autologous bone marrow stem cells-scaffold is superior to traditional treatment in moderate to severe intrauterine adhesion patients in increasing their ongoing pregnancy rate,we conducted this randomized controlled clinical trial.Totally 195 participants with moderate to severe intrauterine adhesion were screened and 152 of them were randomly assigned in a 1:1 ratio to either group with autologous bone marrow stem cells-scaffold plus Foley balloon catheter or group with only Foley balloon catheter(control group)from February 2016 to January 2020.The per-protocol analysis included 140 participants:72 in bone marrow stem cells-scaffold group and 68 in control group.The ongoing pregnancy occurred in 45/72(62.5%)participants in the bone marrow stem cells-scaffold group which was significantly higher than that in the control group(28/68,41.2%)(RR=1.52,95%CI 1.08–2.12,P=0.012).The situation was similar in live birth rate(bone marrow stem cells-scaffold group 56.9%(41/72)vs.control group 38.2%(26/68),RR=1.49,95%CI 1.04–2.14,P=0.027).Compared with control group,participants in bone marrow stem cells-scaffold group showed more menstrual blood volume in the 3rd and 6th cycles and maximal endometrial thickness in the 6th cycle after hysteroscopic adhesiolysis.The incidence of mild placenta accrete was increased in bone marrow stem cells-scaffold group and no severe adverse effects were observed.In conclusion,transplantation of bone marrow stem cells-scaffold into uterine cavities of the participants with moderate to severe intrauterine adhesion increased their ongoing pregnancy and live birth rates,and this therapy was relatively safe.

Construction of functional neural network tissue combining CBD-NT3-modified linear-ordered collagen scaffold and TrkC-modified iPSC-derived neural stem cells for spinal cord injury repair

Induced pluripotent stem cells(iPSCs)can be personalized and differentiated into neural stem cells(NSCs),thereby effectively providing a source of transplanted cells for spinal cord injury(SCI).To further improve the repair efficiency of SCI,we designed a functional neural network tissue based on TrkC-modified iPSC-derived NSCs and a CBD-NT3-modified linear-ordered collagen scaffold(LOCS).We confirmed that transplantation of this tissue regenerated neurons and synapses,improved the microenvironment of the injured area,enhanced remodeling of the extracellular matrix,and promoted functional recovery of the hind limbs in a rat SCI model with complete transection.RNA sequencing and metabolomic analyses also confirmed the repair effect of this tissue from multiple perspectives and revealed its potential mechanism for treating SCI.Together,we constructed a functional neural network tissue using human iPSCs-derived NSCs as seed cells based on the interaction of receptors and ligands for the first time.This tissue can effectively improve the therapeutic effect of SCI,thus confirming the feasibility of human iPSCs-derived NSCs and LOCS for SCI repair and providing a valuable direction for SCI research.

Cu(II)-baicalein enhance paracrine effect and regenerative function of stem cells in patients with diabetes

The development of engineered or modified autologous stem cells is an effective strategy to improve the efficacy of stem cell therapy.In this study,the stemness and functionality of adipose stem cells derived from type 1 diabetic donors(T1DM-ASC)were enhanced by treatment with Cu(II)-baicalein microflowers(Cu-MON).After treatment with Cu-MON,T1DM-ASC showed enhanced expression of the genes involved in the cytokine-cytokine receptor interaction pathway and increased cytokine secretion.Among the top 13 differentially expressed genes between T1DM-ASC and Cu-MON-treated T1DM-ASC(CMTA),some genes were also expressed in HUVEC,Myoblast,Myofibroblast,and Vascular Smooth Muscle cells,inferring the common role of these cell types.In vivo experiments showed that CMTA had the same therapeutic effect as adipose-derived stem cells from non-diabetic donors(ND-ASC)at a 15%cell dose,greatly reducing the treatment cost.Taken together,these findings suggest that Cu-MON promoted angiogenesis by promoting the stemness and functionality of T1DM-ASC and influencing multiple overall repair processes,including paracrine effects.

Transplantation of UC-MSCs on collagen scaffold activates follicles in dormant ovaries of POF patients with long history of infertility

Premature ovarian failure(POF) is a refractory disease for clinical treatment with the goal of restoring fertility. In this study,umbilical cord mesenchymal stem cells on a collagen scaffold(collagen/UC-MSCs) can activate primordial follicles in vitro via phosphorylation of FOXO3 a and FOXO1. Transplantation of collagen/UC-MSCs to the ovaries of POF patients rescued overall ovarian function, evidenced by elevated estradiol concentrations, improved follicular development, and increased number of antral follicles. Successful clinical pregnancy was achieved in women with POF after transplantation of collagen/UC-MSCs or UC-MSCs. In summary, collagen/UC-MSC transplantation may provide an effective treatment for POF.

One-year clinical study of NeuroR egen scaffold implantation following scar resection in complete chronic spinal cord injury patients

The objective of this clinical study was to assess the safety and feasibility of the collagen scaffold, Neuro Regen scaffold, one year after scar tissue resection and implantation. Scar tissue is a physical and chemical barrier that prevents neural regeneration. However, identification of scar tissue is still a major challenge. In this study, the nerve electrophysiology method was used to distinguish scar tissue from normal neural tissue, and then different lengths of scars ranging from 0.5–4.5 cm were surgically resected in five complete chronic spinal cord injury(SCI) patients. The NeuroR egen scaffold along with autologous bone marrow mononuclear cells(BMMCs), which have been proven to promote neural regeneration and SCI recovery in animal models, were transplanted into the gap in the spinal cord following scar tissue resection. No obvious adverse effects related to scar resection or Neuro Regen scaffold transplantation were observed immediately after surgery or at the 12-month follow-up. In addition, patients showed partially autonomic nervous function improvement, and the recovery of somatosensory evoked potentials(SSEP) from the lower limbs was also detected. The results indicate that scar resection and Neuro Regen scaffold transplantation could be a promising clinical approach to treating SCI.

Collagen-binding basic fibroblast growth factor improves functional remodeling of scarred endometrium in uterine infertile women: a pilot study

Intrauterine adhesion(IUA) is a common cause of uterine infertility and one of the most severe clinical features is endometrial fibrosis namely endometrial scarring for which there are few cures currently. Blocked angiogenesis is the main pathological change in the scarred endometrium. The fibroblast growth factor 2(b FGF), a member of FGF family, is usually applied to promote healing of refractory ulcer and contributes to angiogenesis of tissues. In this study, the sustained-release system of b FGF100 μg was administrated around scarred endometrium guiding by ultrasound every 4 weeks in 18 patients(2–4 times). Results showed that after treatment, the menstrual blood volume, endometrial thickness and the scarred endometrial area were improved.Histological study showed blood vessel density increased obviously. Three patients(3/18) achieved pregnancy over 20 gestational weeks. Therefore, administrating the b FGF surrounding scarred endometrium may provide a new therapeutic approach for the patients with endometrial fibrosis.

Transplantation of adult spinal cord grafts into spinal cord transected rats improves their locomotor function

Grafted embryonic central neural tissue pieces can recover function of hemisected spinal cord in neonatal rats and promote axonal growth in adults. However, spinal cord segments from adults have not been used as donor segments for allogeneic transplantation. Here, we utilized adult spinal cord tissue grafts(aSCGs) as donor constructs for repairing complete spinal cord injury(SCI). Moreover, to provide a favourable microenvironment for SCI treatment, a growth factor cocktail containing three growth factors(brain-derived neurotrophic factor, neurotrophin-3 and vascular endothelial growth factor), was applied to the aSCG transplants. We found that the locomotor function was significantly improved 12 weeks after transplantation of aSCGs into the spinal cord lesion site in adult rats. Transplantation of aSCGs combined with these growth factors enhanced neuron and oligodendrocyte survival and functional restoration. These encouraging results indicate that treatment of complete SCI by transplanting aSCGs, especially in the presence of growth factors, has a positive effect on motor functional recovery, and therefore could be considered as a possible therapeutic strategy for SCI.

Single-cell RNA sequencing reveals Nestin+active neural stem cells outside the central canal after spinal cord injury

Neural stem cells(NSCs)in the spinal cord hold great potential for repair after spinal cord injury(SCI).The ependyma in the central canal(CC)region has been considered as the NSCs source in the spinal cord.However,the ependyma function as NSCs after SCI is still under debate.We used Nestin as a marker to isolate potential NSCs and their immediate progeny,and characterized the cells before and after SCI by single-cell RNA-sequencing(scRNA-seq).We identified two subgroups of NSCs:the subgroup located within the CC cannot prime to active NSCs after SCI,while the subgroup located outside the CC were activated and exhibited the active NSCs properties after SCI.We demonstrated the comprehensive dynamic transcriptome of NSCs from quiescent to active NSCs after SCI.This study reveals that Nestin+cells outside CC were NSCs that activated upon SCI and may thus serve as endogenous NSCs for regenerative treatment of SCI in the future.

Long-term clinical observation of patients with acute and chronic complete spinal cord injury after transplantation of Neuro Regen scaffold

Spinal cord injury(SCI)often results in an inhibitory environment at the injury site.In our previous studies,transplantation of a scaffold combined with stem cells was proven to induce neural regeneration in animal models of complete SCI.Based on these preclinical studies,collagen scaffolds loaded with the patients’own bone marrow mononuclear cells or human umbilical cord mesenchymal stem cells were transplanted into SCI patients.Fifteen patients with acute complete SCI and 51 patients with chronic complete SCI were enrolled and followed up for 2 to 5 years.No serious adverse events related to functional scaffold transplantation were observed.Among the patients with acute SCI,five patients achieved expansion of their sensory positions and six patients recovered sensation in the bowel or bladder.Additionally,four patients regained voluntary walking ability accompanied by reconnection of neural signal transduction.Among patients with chronic SCI,16 patients achieved expansion of their sensation level and 30 patients experienced enhanced reflexive defecation sensation or increased skin sweating below the injury site.Nearly half of the patients with chronic cervical SCI developed enhanced finger activity.These long-term follow-up results suggest that functional scaffold transplantation may represent a feasible treatment for patients with complete SCI.

The Three-Dimensional Collagen Scaffold Improves the Sternness of Rat Bone Marrow Mesenchymal Stem Cells

Mesenchymal stem cells （MSCs） show the great promise for the treatment of a variety of diseases because of their self-renewal and multipotential abilities. MSCs are generally cultured on two-dimensional （2D） substrate in vitro. There are indications that they may simultaneously lose their sternness and multipotentiality as the result of prolonged 2D culture. In this study, we used three-dimensional （3D） collagen scaffolds as rat MSCs cartier and compared the properties of MSCs on 3D collagen scaffolds with monolayer cultured MSCs. The results demonstrated that collagen scaffolds were suitable for rat MSCs adherence and proliferation. More importantly, compared to MSCs under 2D culture, 3D MSCs significantly maintained higher expression levels of stemness genes （Oct4, Sox2, Rex-1 and Nanog）, yielded high frequencies of colony-forming units-fibroblastic （CFU-F） and showed enhanced osteogenic and adipogenic differentiation efficiency upon induction. Thus, 3D collagen scaffolds may be beneficial for expanding rat MSCs while maintaining the stem cell properties in vitro.

The novel OCT4 spliced variant OCT4B1 can generate three protein isoforms by alternative splicing into OCT4B

OCT4 is one of the key transcription factors in maintaining the pluripotency and self-renewal of embryonic stem （ES） cells.Human OCT4 can generate two isoforms OCT4A and OCT4B by alternative splicing.OCT4B1 is a recently discovered novel OCT4 spliced variant,which has been considered as a putative marker of stemness.Compared with the OCT4B mRNA,OCT4B1 mRNA is generated by retaining intron 2 as a cryptic exon which contains a TGA stop codon in it.As a result,the protein product of OCT4B1 mRNA could be truncated.Interestingly,we present here that OCT4B1 can indirectly produce the same protein products as OCT4B.We have demonstrated that OCT4B1 mRNA can be spliced into OCT4B mRNA,and encode three protein isoforms.The splicing of OCT4B1 mRNA into OCT4B mRNA can be remarkably inhibited by the mutation of the classical splicing site.Our result suggests that OCT4B mRNA may originate from OCT4B1 mRNA by alternative splicing.

Microvascular endothelial cells derived from spinal cord promote spinal cord injury repair

Neural regeneration after spinal cord injury (SCI) closely relates to the microvascular endothelial cell (MEC)- mediated neurovascular unit formation. However, the effects of central nerve system-derived MECs on neovascularization and neurogenesis, and potential signaling involved therein, are unclear. Here, we established a primary spinal cord-derived MECs (SCMECs) isolation with high cell yield and purity to describe the differences with brain-derived MECs (BMECs) and their therapeutic effects on SCI. Transcriptomics and proteomics revealed differentially expressed genes and proteins in SCMECs were involved in angiogenesis, immunity, metabolism, and cell adhesion molecular signaling was the only signaling pathway enriched of top 10 in differentially expressed genes and proteins KEGG analysis. SCMECs and BMECs could be induced angiogenesis by different stiffness stimulation of PEG hydrogels with elastic modulus 50-1650 Pa for SCMECs and 50-300 Pa for BMECs, respectively. Moreover, SCMECs and BMECs promoted spinal cord or brain-derived NSC (SNSC/BNSC) proliferation, migration, and differentiation at different levels. At certain dose, SCMECs in combination with the NeuroRegen scaffold, showed higher effectiveness in the promotion of vascular reconstruction. The potential underlying mechanism of this phenomenon may through VEGF/AKT/eNOS- signaling pathway, and consequently accelerated neuronal regeneration and functional recovery of SCI rats compared to BMECs. Our findings suggested a promising role of SCMECs in restoring vascularization and neural regeneration.

Allotransplantation of adult spinal cord tissues after complete transected spinal cord injury: long-term survival and functional recovery in canines

Spinal cord injury(SCI), especially complete transected SCI, leads to loss of cells and extracellular matrix and functional impairments. In a previous study, we transplanted adult spinal cord tissues(aSCTs) to replace lost tissues and facilitate recovery in a rat SCI model. However, rodents display considerable differences from human patients in the scale, anatomy and functions of spinal cord systems, and responses after injury. Thus, use of a large animal SCI model is required to examine the repair efficiency of potential therapeutic approaches. In this study, we transplanted allogenic aSCTs from adult dogs to the lesion area of canines after complete transection of the thoracic spinal cord, and investigated the long-term cell survival and functional recovery. To enhance repair efficiency, a growth factor cocktail was added during aSCT transplantation, providing a favorable microenvironment. The results showed that transplantation of a SCTs, in particular with the addition of growth factors, significantly improves locomotor function restoration and increases the number of neurofilament-, microtubule-associated protein2-, 5-hydroxytryptamine-, choline acetyltransferase-and tyrosine hydroxylase-positive neurons in the lesion area at 6 months post-surgery. In addition, we demonstrated that donor neurons in a SCTs can survive for a long period after transplantation. This study showed for the first time that transplanting aSCTs combined with growth factor supplementation facilitates reconstruction of injured spinal cords, and consequently promotes long lasting motor function recovery in a large animal complete transected SCI model, and therefore could be considered as a possible therapeutic strategy in humans.

Lineage tracing reveals the origin of Nestin-positive cells are heterogeneous and rarely from ependymal cells after spinal cord injury

Nestin is expressed extensively in neural stem/progenitor cells during neural development, but its expression is mainly restricted to the ependymal cells in the adult spinal cord. After spinal cord injury(SCI), Nestin expression is reactivated and Nestinpositive(Nestin;) cells aggregate at the injury site. However, the derivation of Nestin;cells is not clearly defined. Here, we found that Nestin expression was substantially increased in the lesion edge and lesion core after SCI. Using a tamoxifen inducible CreER(T2)-loxP system, we verified that ependymal cells contribute few Nestin;cells either to the lesion core or the lesion edge after SCI. In the lesion edge, GFAP+astrocytes were the main cell type that expressed Nestin;they then formed an astrocyte scar.In the lesion core, Nestin;cells expressed αSMA or Desmin, indicating that they might be derived from pericytes. Our results reveal that Nestin;cells in the lesion core and edge came from various cell types and rarely from ependymal cells after complete transected SCI, which may provide new insights into SCI repair.

Injectable collagen scaffold promotes swine myocardial infarction recovery by long-term local retention of transplanted human umbilical cord mesenchymal stem cells

Stem cell therapy is an attractive approach for recovery from myocardial infarction(MI)but faces the challenges of rapid diffusion and poor survival after transplantation.Here we developed an injectable collagen scaffold to promote the long-term retention of transplanted cells in chronic MI.Forty-five minipigs underwent left anterior descending artery(LAD)ligation and were equally divided into three groups 2 months later(collagen scaffold loading with human umbilical mesenchymal stem cell(hUMSC)group,hUMSC group,and placebo group(only phosphate-buffered saline(PBS)injection)).Immunofluorescence staining indicated that the retention of transplanted cells was promoted by the collagen scaffold.Echocardiography and cardiac magnetic resonance imaging(CMR)showed much higher left ventricular ejection fraction(LVEF)and lower infarct size percentage in the collagen/hUMSC group than in the hUMSC and placebo groups at 12 months after treatment.There were also higher densities of vWf-,α-sma-,and cTnT-positive cells in the infarct border zone in the collagen/cell group,as revealed by immunohistochemical analysis,suggesting better angiogenesis and more cardiomyocyte survival after MI.Thus,the injectable collagen scaffold was safe and effective on a large animal myocardial model,which is beneficial for constructing a favorable microenvironment for applying stem cells in clinical MI.

Transplantation of collagen scaffold with autologous bone marrow mononuclear cells promotes functional endometrium reconstruction via downregulating ΔNp63 expression in Asherman＇s syndrome

Asherman's syndrome(AS) is a common disease that presents endometrial regeneration disorder. However, little is known about its molecular features of this aregenerative endometrium in AS and how to reconstruct the functioning endometrium for the patients with AS. Here, we report that ΔNp63 is significantly upregulated in residual epithelial cells of the impaired endometrium in AS; the upregulated-ΔNp63 induces endometrial quiescence and alteration of stemness. Importantly, we demonstrate that engrafting high density of autologous bone marrow mononuclear cells(BMNCs) loaded in collagen scaffold onto the uterine lining of patients with AS downregulates ΔNp63 expression, reverses ΔNp63-induced pathological changes, normalizes the stemness alterations and restores endometrial regeneration. Finally, five patients achieved successful pregnancies and live births. Therefore, we conclude that ΔNp63 is a crucial therapeutic target for AS. This novel treatment significantly improves the outcome for the patients with severe AS.

Recent developments in regenerative ophthalmology

Regenerative medicine(RM)is one of the most promising disciplines for advancements in modern medicine,and regenerative ophthalmology(RO)is one of the most active fields of regenerative medicine.This review aims to provide an overview of regenerative ophthalmology,including the range of tools and materials being used,and to describe its application in ophthalmologic subspecialties,with the exception of surgical implantation of artificial tissues or organs(e.g.,contact lens,artificial cornea,intraocular lens,artificial retina,and bionic eyes)due to space limitations.In addition,current challenges and limitations of regenerative ophthalmology are discussed and future directions are highlighted.

Human placenta-derived mesenchymal stem cells loaded on linear ordered collagen scaffold improves functional recovery after completely transected spinal cord injury in canine

Traumatic spinal cord injury(SCI) is a major challenge in the clinic. In this study, we sought to examine the synergistic effects of linear ordered collagen scaffold(LOCS) and human placenta-derived mesenchymal stem cells(hPMSCs) when transplanted into completely transected beagle dogs. After 36 weeks observation, we found that LOCS+hPMSCs implants promoted better hindlimb locomotor recovery than was observed in the non-treatment(control) group and LOCS group. Histological analysis showed that the regenerated tissue after treatment was well integrated with the host tissue, and dramatically reduced the volume of cystic and chondroitin sulfate proteoglycans(CSPGs) expression. Furthermore, the LOCS+hPMSCs group also showed more neuron-specific βIII-tubulin(Tuj-1)-and NeuN-positive neurons in the lesion area, as well as axonal regeneration, remyelination and synapse formation in the lesion site. Additionally, dogs in the LOCS+hPMSCs group experienced enhanced sprouting of both ascending(CGRP-positive) sensory fibers and descending(5-HT-and TH-positive) motor fibers at the lesion area. All these data together suggested that the combined treatment had beneficial effects on neuronal regeneration and functional improvement in a canine complete transection model. Therefore, LOCS+hPMSCs implantation holds a great promise for bridging the nerve defect and may be clinically useful in the near future.

Complete canine spinal cord transection model: a large animal model for the translational research of spinal cord regeneration

Traumatic spinal cord injury (SCI) usually results in devastating neurologic deficits and disability. In the United States,approximately 12,500 new cases are reported each year, while an estimated 100,000–140,000 new cases occur every year in China (National Spinal Cord Injury Statistical Center, 2016).Spinal cord injuries are highly disabling and primarily affect young adults, and therefore create great psychological and financial burden on the affected individuals and their families.