低氧环境和生长分化因子-5联合诱导促进人骨髓基质干细胞分化成软骨细胞的研究

目的 探讨低氧环境下生长分化因子-5 (GDF-5)诱导人骨髓基质干细胞(hBMSCs)“自组装”形成工程化软骨的可行性和有效性。方法 分离培养hBMSCs,流式细胞学方法鉴定。将hBMSCs用含100 ng/mL GDF-5软骨诱导液(CM)分别在低氧（A组）和正常氧（B组）环境下诱导培养3周,RT-PCR检测Ⅱ型胶原和Aggrecan表达情况。将A、B两组hBMSCs消化后,按一定密度接种于2％琼脂糖包被的24孔板,在不同氧浓度下CM继续诱导3周,免疫组化法检测组织Ⅰ、Ⅱ型胶原表达,甲苯胺蓝染色检测葡萄糖胺聚糖(GAG)表达。结果 hBMSCs呈梭形漩涡状生长,高表达CD44、CD29,不表达CD45分子。诱导5d后A组细胞较B组明显增殖,诱导10dA组细胞体积较B组小。含GDF-5的CM诱导hBMSCs 3周后,Ⅱ型胶原和Aggrecan mRNA表达阳性,A组Ⅱ型胶原和Aggrecan表达量较B组均明显增加,差异有统计学意义(P＜0.05)。GDF-5诱导hBMSCs可“自组装”形成一定形状大小类软骨样组织,A组Ⅱ型胶原表达较B组增加,A组Ⅰ型胶原表达量下降,B组表达阳性;A组甲苯胺蓝染色异染加深。结论 低氧促进GDF-5诱导hBMSCs向软骨分化,低氧环境下GDF-5诱导软骨分化的hBMSCs“自组装”形成的工程化软骨更具有软骨表型。     

Use of Allogeneic Hypoxic Mesenchymal Stem Cells For Treating Disc Degeneration in Rabbits

Intervertebral discs (IVDs) are important biomechanical components of the spine. Once degenerated, mesenchymal stem cell (MSC)‐based therapies may aid in the repair of these discs. Although hypoxic preconditioning enhances the chondrogenic potential of MSCs, it is unknown whether bone marrow MSCs expanded under hypoxic conditions (1% O₂, here referred to as hypoxic MSCs) are better than bone marrow MSCs expanded under normoxic conditions (air, here referred to as normoxic MSCs) with regards to disc regeneration capacity. The purpose of this study was to compare the therapeutic effects of hypoxic and normoxic MSCs in a rabbit needle puncture degenerated disc model after intra‐disc injection. Six weeks after needle puncture, MSCs were injected into the IVD. A vehicle‐treated group and an un‐punctured sham‐control group were included as controls. The tissues were analyzed by histological and immunohistochemical methods 6 and 12 weeks post‐injection. At 6 and 12 weeks, less disc space narrowing was evident in the hypoxic MSC‐treated group compared to the normoxic MSC‐treated group. Significantly better histological scores were observed in the hypoxic MSC group. Discs treated with hypoxic MSCs also demonstrated significantly better extracellular matrix deposition in type II and XI collagen. Increased CD105 and BMP‐7 expression were also observed upon injection of hypoxic MSCs. In conclusion, hypoxic MSC injection was more effective than normoxic MSC injection for reducing IVD degeneration progression in vivo. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1440–1450, 2019.     

利用人骨髓基质干细胞异位构建组织工程化骨的实验研究

目的 探讨以人骨髓基质干细胞为种子细胞、以部分脱钙骨为支架材料在体内构建组织工程化骨的可行性及其成骨机制。方法 培养、扩增人骨髓基质干细胞，将第4代hBMSCs接种于部分脱钙骨支架上，通过扫描电镜观察其生长和粘附情况，并测量粘附率。于裸鼠皮下植入人骨髓基质干细胞和部分脱钙骨复合物，以无细胞部分脱钙骨作对照。8及12周取材观察。结果 8及12周复合物植入组均见新骨形成，多数骨小梁表面衬有一层成骨细胞样细胞，提示可能存在膜内成骨。单纯部分脱钙骨植入组未见新骨形成。结论 人骨髓基质干细胞与部分脱钙骨复合可以在体内构建组织工程化骨；其成骨机制可能为膜内成骨。     

人骨髓间充质干细胞体外成骨分化过程中成骨相关基因的动态表达

目的：系统研究人骨髓间充质干细胞（hBMSCs）体外成骨诱导分化过程中成骨相关基因的表达变化。方法：应用密度梯度离心法分离hBMSCs,取第2代细胞通过流式检测及多向诱导分化方法进行干细胞鉴定;应用RT-PCR法对hBMSCs在体外成骨诱导不同时间点的成骨相关基因表达进行检测。结果：第2代hBMSCs表达间充质干细胞表面标志CD44、CD90,具有成脂和成骨分化潜能。成骨相关基因在诱导早期部分表达,中期均有表达,基因表达大部分在14天达高峰,与矿化相关的基因表达在21天达高峰。结论：hBMSCs体外成骨诱导过程中成骨相关基因呈动态表达,其表达时序与成骨细胞生理发育基本相似。     

Effects of hypoxia on differentiation from human placenta-derived mesenchymal stem cells to nucleus pulposus-like cells

Background context

Low back pain is a frequently occurring disease caused by intervertebral disc degeneration. Mesenchymal stem cells (MSCs) are a possible treatment modality. Studies have shown MSCs can be transformed into nucleus pulposus-like cells under normoxic conditions. However, this is not a true representation of the hypoxic environment nucleus pulposus cells experience during in vivo growth and differentiation.

Purpose

To determine the effects of a hypoxic environment on the differentiation of human placenta-derived mesenchymal stem cells (PMSCs) to nucleus pulposus-like cells.

Study design

An experimental study.

Methods

Placenta-derived mesenchymal stem cells were cultured and the mesenchymal lineage was confirmed by flow cytometry. Two groups of PMSCs were then cultured under different oxygen concentrations creating a hypoxic group and normoxic group. The proliferation of cells in each group was compared by cell counting kit-8 on Day 1, 3, 5, and 7. Real-time polymerase chain reaction on Days 3 and 7 compared the expressions of Sox-9, Type II collagen, aggrecan, and hypoxia inducible factor-1α (HIF-1α) between the two groups. Immunofluorescence was used to compare the expression of Type II collagen between the two groups after 14 days.

Results

Placenta-derived mesenchymal stem cells were successfully isolated and cultured. Mesenchymal markers were positive. On Days 3 and 5, the hypoxic group had a significantly higher proliferation rate than the normoxic group (p<.05). The expression of Sox-9 and HIF-1α was significantly higher (p<.05) in the hypoxic group at Days 3 and 7. Type II collagen and aggrecan expressions were significantly higher (p<.05) in the hypoxic group at Day 7. The hypoxic group stained more positive for Type II collagen at Day 14.

Conclusions

Hypoxic conditions lead to an increased differentiation and proliferation of nucleus pulposus-like cells. Placenta-derived mesenchymal stem cells cultured in nucleus pulposus inducing media and a hypoxic environment show enhanced expression of the nucleus pulposus-like cell markers, Sox-9, Type II collagen, aggrecan, and HIF-1α.     

人骨髓间充质干细胞分离培养及体外诱导分化为成骨细胞

[目的]建立人骨髓来源的间充质干细胞( hBMSCs)分离、培养及传代的方法,观察成hBMSCs体外成骨潜能.[方法]采用全骨髓贴壁筛选法分离培养hBMSCs,流式细胞仪检测细胞表型;所得细胞第3代用含100 nmol/L地塞米松、5mmol/Lβ -甘油磷酸钠,50 μg/ml抗坏血酸的条件培养基进行骨诱导,茜素红染色鉴定.[结果]分离培养的细胞流式细胞术检测显示CD44、CD105阳性,而CD34、CD45阴性,符合MSCs特征;hBMSCs经骨诱导后可形成钙结节,茜素红染色显示阳性.[结论]全骨髓贴壁筛选法可分离获得高纯度的hBMSCs,其在体外具有成骨潜能.     

Sequential differentiation of mesenchymal stem cells in an agarose scaffold promotes a physis‐like zonal alignment of chondrocytes

Chondrocytes of the epiphyseal growth plate (physis) differentiate and mature in defined linear zones. The current study examines the differentiation of human bone marrow derived mesenchymal stem cells (hBMSCs) into zonal physeal cartilage. hBMSCs were embedded in an agarose scaffold with only the surface of the scaffold in direct contact with the culture medium. The cells were differentiated using a two‐step system involving the sequential addition of TGFβ followed by BMP2. The resultant samples displayed a heterogenic population of physis‐like collagen type 2 positive cells including proliferating chondrocytes and mature chondrocytes showing hypertrophy, expression of early bone markers and matrix mineralization. Histological analysis revealed a physis‐like linear zonal alignment of chondrocytes in varying stages of differentiation. The less mature chondrocytes were seen at the base of the construct while hypertrophic chondrocytes and matrix mineralization was observed closer to the surface of the construct. The described differentiation protocol using hBMSCs in an agarose scaffold can be used to study the factors and conditions that influence the differentiation, proliferation, maturation, and zonal alignment of physeal chondrocytes. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1753–1759, 2012     

骨髓基质干细胞及其应用研究进展

骨髓基质干细胞(BMSCs)作为一种干细胞，由于具有很强的自我更新能力和多分化潜能，近年来引起了广泛的注意，体内外试验中已发现BMSCs可以分化为骨组织和软骨组织、神经组织等组织的细胞，并且具有修复以上各组织缺损的能力；同时发现BMSCs是一种理想的基因载体，从而达到基因治疗的目的。本文旨在对BMSCs的分离、培养、生物学特性、基因治疗及其组织修复中的应用研究进展作一综述。     

Human adipose-derived stem cells: Potential clinical applications in surgery

Regenerative medicine is emerging as a rapidly evolving field of research and therapeutics. Stem cells hold great promise for future translational research and clinical applications in many fields. Much research has focused on mesenchymal stem cells isolated from bone marrow in vitro and in vivo; however, bone marrow procurement causes considerable discomfort to the patient and yields a relatively small number of harvested cells. By contrast, adipose tissue represents an abundant and easily accessible source of adult stem cells, termed adipose-derived stem cells (ADSCs), with the ability to equally differentiate along multiple lineage pathways. These stem cells have angiogenic properties, possibly because of their secretion of cytokines. They may also play a role in healing acute and chronic tissue damage. Subsequently, they have a wide range of potential clinical implications. This article reviews the potential preclinical and clinical applications of mesenchymal stem cells, especially ADSCs, in surgery.     

hBMP-2基因改良修饰骨髓间充质干细胞和人脐血间充质干细胞研究

目的研究hBMP-2基因改良修饰骨髓和人脐血间充质干细胞的方法,并比较修饰结果。 方法联合应用密度梯度离心法和贴壁培养法分离、培养骨髓和人脐血间充质干细胞,通过高效非脂质体试剂X-treme GENE介导重组hBMP-2质粒转染骨髓和人脐血间充质干细胞,倒置荧光显微镜检测荧光强度并计算转染效率,qPCR检测hBMP-2及软骨连接蛋白在两种细胞中的表达,免疫组化检测细胞中Ⅱ型胶原的变化。 结果成功分离、培养出骨髓和人脐血间充质干细胞,两种细胞具有不同的生长特性。高效非脂质体试剂介导的重组hBMP-2质粒成功转染骨髓和人脐血间充质干细胞,转染骨髓间充质干细胞效率[（18.44±5.94）%]低于人脐血间充质干细胞[（27.74±7.59）%],且差异有统计学意义（t=3.027,P<0.05）。转染后的两种细胞均检测到hBMP-2、软骨连接蛋白及Ⅱ型胶原的表达。 结论hBMP-2基因可以有效改良修饰骨髓和人脐血间充质干细胞,且能促进其向软骨细胞方向分化。     

Metabolic Switch Under Glucose Deprivation Leading to Discovery of NR2F1 as a Stimulus of Osteoblast Differentiation

Poor survival of grafted cells is the major impediment of successful cell-based therapies for bone regeneration. Implanted cells undergo rapid death in an ischemic environment largely because of hypoxia and metabolic stress from glucose deficiency. Understanding the intracellular metabolic processes and finding genes that can improve cell survival in these inhospitable conditions are necessary to enhance the success of cell therapies. Thus, the purpose of this study was to investigate changes of metabolic profile in glucose-deprived human bone marrow stromal/stem cells (hBMSCs) through metabolomics analysis and discover genes that could promote cell survival and osteogenic differentiation in a glucose-deprived microenvironment. Metabolomics analysis was performed to determine metabolic changes in a glucose stress metabolic model. In the absence of glucose, expression levels of all metabolites involved in glycolysis were significantly decreased than those in a glucose-supplemented state. In glucose-deprived osteogenic differentiation, reliance on tricarboxylic acid cycle (TCA)-predicted oxidative phosphorylation instead of glycolysis as the main mechanism for energy production in osteogenic induction. By comparing differentially expressed genes between glucose-deprived and glucose-supplemented hBMSCs, NR2F1 (Nuclear Receptor Subfamily 2 Group F Member 1) gene was discovered to be associated with enhanced survival and osteogenic differentiation in cells under metabolic stress. Small, interfering RNA (siRNA) for NR2F1 reduced cell viability and osteogenic differentiation of hBMSCs under glucose-supplemented conditions whereas NR2F1 overexpression enhanced osteogenic differentiation and cell survival of hBMSCs in glucose-deprived osteogenic conditions via the protein kinase B (AKT)/extracellular signal-regulated kinase (ERK) pathway. NR2F1-transfected hBMSCs significantly enhanced new bone formation in a critical size long-bone defect of rats compared with control vector-transfected hBMSCs. In conclusion, the results of this study provide an understanding of the metabolic profile of implanted cells in an ischemic microenvironment and demonstrate that NR2F1 treatment may overcome this deprivation by enhancing AKT and ERK regulation. These findings can be utilized in regenerative medicine for bone regeneration. © 2022 American Society for Bone and Mineral Research (ASBMR).     

Enhanced wound-healing quality with bone marrow mesenchymal stem cells autografting after skin injury

Adult stem cells exist in various tissues and organs and have the potential to differentiate into different cell lineages, including bone, cartilage, fat, tendon, muscle, and epithelial cells of the gastrointestinal tract. Here, we report that the in vitro expanded and purified bone marrow mesenchymal stem cells (MSCs) might take on phenotypes with characteristics of vascular endothelial cells (7% on day 3 and 15% on day 1) or epidermal cells (3% on day 3 and 13% on day 1) after being cultured under different lineage-specific culture conditions. Also, in vivo grafting experiments showed that 5-bromodeoxyuridine-labeled MSCs could convert into the phenotypes of vascular endothelial cells (3.43, 3.46, and 2.94% on days 7, 14, and 28, respectively) in granulation tissues, sebaceous duct cells, and epidermal cells (0-1.49%) in regenerated skin, implying that these grafted MSCs might have transdifferentiated into the above three cell types. Animal autografting experiments with MSCs further confirmed that indices pertaining to wound healing quality, such as the speed of reepithelialization, the number of epidermal ridges and thickness of the regenerated epidermis, the morphology and the number and arrangement of microvasculature, fibroblasts and collagen, were much enhanced. Our results indicate that locally delivered bone marrow MSCs can enhance wound healing quality, and may generate de novo intact skin, resulting in perfect skin regeneration after full-thickness injury.     

Stammzellen aus dem Fettgewebe

While stem cells derived from the bone marrow are well-known in clinical medicine, fatty tissue as a source of mesenchymal stem cells is still the subject of recent research. However, adipose-derived stem cells (ASC) are not only harvested less invasively, i.e. via minimally invasive liposuction, but also yield higher numbers of multipotent stem cells. Due to cell-cell interactions and also because of the very favorable secretion profile of growth factors and cytokines ASCs displayed an extraordinary regenerative potential in recent preclinical and clinical applications and achieved a significantly better healing in ischemic muscle, heart, and brain insults and in impaired wound healing. ASCs enhanced regeneration in skeletal tissues such as cartilage or bone. They also revealed immunomodulatory effects and improved the clinical status in immunological diseases. In conclusion ASCs are comparable to bone marrow-derived stem cells concerning possible applications in clinical medicine.     

Scale‐up of MSC under hypoxic conditions for allogeneic transplantation and enhancing bony regeneration in a rabbit calvarial defect model

To realize the therapeutic potential of mesenchymal stem cells (MSCs), we aimed to develop a method for isolating and expanding New Zealand rabbit MSCs in a great scale. Rabbit MSCs expanded under hypoxic and normoxic conditions were compared in terms of replication capacity, differentiation potential, and the capacity for allogeneic transplantation in a calvarial defect model. The cells from all tested rabbits were expanded more rapidly when plated at low‐density under hypoxic conditions compared to under normoxic conditions. Moreover, cells expanded under hypoxic conditions increased in the potential of osteoblastic, adipocytic, and chondrocytic differentiation. More importantly, radiographic analysis and micro‐CT measurement of bone volume revealed the hypoxic cells when transplanted in the calvarial defects of another rabbit increased in the ability to repair bone defect compared to the normoxic cells. Six weeks after allogeneic transplantation of hypoxic MSCs, histological analysis revealed a callus spanned the length of the defect, and several bone tissues spotted in the implant. At 12 weeks, new bone had formed throughout the implant. Using BrdU labeling to track the transplanted cells, the hypoxic cells were more detected in the newly formed bone compared to the normoxic cells. For defects treated with allogeneic MSCs, no adverse host response could be detected at any time‐point. In conclusion, we have developed a robust method for isolation and expansion of rabbit MSCs by combining low‐density with hypoxic culture, which can be applied for the design of clinical trials in allogeneic transplantation of MSCs for bone healing. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1213–1220, 2012     

体外转染GFP-Bcl-2基因对低氧条件下间充质干细胞细胞周期的影响

目的探讨低氧条件下诱导骨髓来源的间充质干细胞（MSCs）向目标细胞分化为相关疾病提供了潜在的临床治疗途径。方法将表达GFP-Bcl-2的慢病毒载体转染大鼠骨髓MSCs使其过量表达（GFP-Bcl-2-MSCs）。在低氧环境下诱导其分化并检测细胞凋亡和增殖。采用流式细胞术检测MSCs和GFP-Bcl-2-MSCs在低氧条件下MSCs细胞细胞周期分布。采用Western blot检测不同组细胞cyclinD1、cyclinE及PCNA表达变化。结果在体外低氧诱导条件下,Bcl-2基因对MSCs有明显的抗凋亡功能。与空载体对照组相比：实验组（GFP-Bcl-2-MSCs）细胞凋亡率下降27%,细胞增殖率升高了58%。但细胞周期分布及cyclinD1、cyclinE、及PCNA的表达无明显差异。结论研究证实了抗凋亡基因修饰的MSCs（GFP-Bcl-2-MSCs）在低氧环境下可抑制细胞凋亡,但细胞周期的相关机制需要进一步研究。     

纳米磁铁颗粒标记人骨髓基质干细胞的初步研究

目的 研究纳米磁性氧化铁颗粒与人骨髓基质干细胞(Human bone marrow stromal cells,hBMSCs)共培养对细胞的生长和分化影响,以评价颗粒的生物相容性,并探讨其作为组织工程种子细胞标记物的可行性.方法 制备两种粒径(6 nm和200 nm)的纳米级超顺磁性氧化铁颗粒,分别在细胞接种时及接种后24 h加入.检测纳米磁铁颗粒和骨髓基质干细胞共培养时细胞的增殖能力和成骨分化能力.结果 纳米磁铁颗粒的种类及加入方式对细胞增殖能力没有明显影响.hBMSCs和6 nm粒径的铁颗粒共培养时能保持较好的ALP活性表达,而200 nm粒径的铁颗粒显著降低了hBMSCs的ALP活性.结论 6 nm粒径的磁性氧化铁颗粒和hBMSCs共培养时,对细胞的增殖和成骨诱导能力没有影响,表明其具有良好的生物相容性,有望成为组织工程种子细胞的标记物来进行磁共振成像(MRI)示踪.     

Human bone marrow stromal cells are efficiently transduced by vesicular stomatitis virus-pseudotyped retrovectors without affecting subsequent osteoblastic differentiation

This study tested the transduction efficiency of human bone marrow stromal cells (hBMSCs) with vesicular stomatitis virus (VSV)-pseudotyped retrovectors and their subsequent osteogenic differentiation in vitro. Two different retrovectors encoding beta-galactosidase (beta-gal) or enhanced green fluorescent protein (eGFP) as marker genes were examined for transduction of hBMSCs. hBMSCs were obtained from bone marrow filtrates of normal donors (aged 5-35 years), cultured in alpha-minimal essential medium (alpha-MEM) containing 10% fetal calf serum and infected with retrovectors soon after the adherent cells started to form individual colonies. Transduced hBMSCs were observed to express eGFP protein 4-7 days after infection in primary cultures, and the majority of hBMSCs were eGFP-positive. hBMSCs were also stained for beta-gal in the secondary cultures and virtually all hBMSCs expressed beta-gal activity. Transduced hBMSCs were examined for their osteogenic potential. These cells were found to express markers of osteogenic differentiation, including alkaline phosphatase, type I collagen, bone sialoprotein, decorin, and osteocalcin, as strongly as uninfected control cells. Mineralization was also induced by dexamethasone in transduced cells as well as control cells. These results demonstrate that hBMSCs are highly susceptible to infection with VSV-pseudotyped retrovectors with the majority of cultured cells expressing the viral transgenes without antibiotic selection. Transduced cells retain their osteogenic potential in vitro. hBMSCs are a promising cellular vehicle for systemic human gene therapy and VSV-pseudotyped retrovectors should be effective for their in vitro transduction prior to cellular engraftment.     

MT1‐MMP and Type II Collagen Specify Skeletal Stem Cells and Their Bone and Cartilage Progeny

Skeletal formation is dependent on timely recruitment of skeletal stem cells and their ensuing synthesis and remodeling of the major fibrillar collagens, type I collagen and type II collagen, in bone and cartilage tissues during development and postnatal growth. Loss of the major collagenolytic activity associated with the membrane‐type 1 matrix metalloproteinase (MT1‐MMP) results in disrupted skeletal development and growth in both cartilage and bone, where MT1‐MMP is required for pericellular collagen dissolution. We show here that reconstitution of MT1‐MMP activity in the type II collagen‐expressing cells of the skeleton rescues not only diminished chondrocyte proliferation, but surprisingly, also results in amelioration of the severe skeletal dysplasia associated with MT1‐MMP deficiency through enhanced bone formation. Consistent with this increased bone formation, type II collagen was identified in bone cells and skeletal stem/progenitor cells of wildtype mice. Moreover, bone marrow stromal cells isolated from mice expressing MT1‐MMP under the control of the type II collagen promoter in an MT1‐MMP‐deficient background showed enhanced bone formation in vitro and in vivo compared with cells derived from nontransgenic MT1‐MMP‐deficient littermates. These observations show that type II collagen is not stringently confined to the chondrocyte but is expressed in skeletal stem/progenitor cells (able to regenerate bone, cartilage, myelosupportive stroma, marrow adipocytes) and in the chondrogenic and osteogenic lineage progeny where collagenolytic activity is a requisite for proper cell and tissue function.     

Ex vivo allotransplantation engineering: Delivery of mesenchymal stem cells prolongs rejection‐free allograft survival

Current pharmacologic regimens in transplantation prevent allograft rejection through systemic recipient immunosuppression but are associated with severe morbidity and mortality. The ultimate goal of transplantation is the prevention of allograft rejection while maintaining recipient immunocompetence. We hypothesized that allografts could be engineered ex vivo (after allotransplant procurement but before transplantation) by using mesenchymal stem cell–based therapy to generate localized immunomodulation without affecting systemic recipient immunocompetence. To this end, we evaluated the therapeutic efficacy of bone marrow–derived mesenchymal stem cells in vitro and activated them toward an immunomodulatory fate by priming in inflammatory or hypoxic microenvironments. Using an established rat hindlimb model for allotransplantation, we were able to significantly prolong rejection‐free allograft survival with a single perioperative ex vivo infusion of bone marrow–derived mesenchymal stem cells through the allograft vasculature, in the absence of long‐term pharmacologic immunosuppression. Critically, transplanted rats rejected a second, nonengineered skin graft from the same donor species to the contralateral limb at a later date, demonstrating that recipient systemic immunocompetence remained intact. This study represents a novel approach in transplant immunology and highlights the significant therapeutic opportunity of the ex vivo period in transplant engineering.