MicroRNAs Modulate Signaling Pathways in Osteogenic Differentiation of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) have been identified in many adult tissues and they have been closely studied in recent years, especially in view of their potential use for treating diseases and damaged tissues and organs. MSCs are capable of self-replication and differentiation into osteoblasts and are considered an important source of cells in tissue engineering for bone regeneration. Several epigenetic factors are believed to play a role in the osteogenic differentiation of MSCs, including microRNAs (miRNAs). MiRNAs are small, single-stranded, non-coding RNAs of approximately 22 nucleotides that are able to regulate cell proliferation, differentiation and apoptosis by binding the 3′ untranslated region (3′-UTR) of target mRNAs, which can be subsequently degraded or translationally silenced. MiRNAs control gene expression in osteogenic differentiation by regulating two crucial signaling cascades in osteogenesis: the transforming growth factor-beta (TGF-β)/bone morphogenic protein (BMP) and the Wingless/Int-1(Wnt)/β-catenin signaling pathways. This review provides an overview of the miRNAs involved in osteogenic differentiation and how these miRNAs could regulate the expression of target genes.     

骨髓间充质干细胞对大鼠胰腺损伤组织的修复作用

目的观察骨髓间充质干细胞(MSCs)对大鼠胰腺损伤组织的修复作用。方法应用贴壁法分离、纯化、扩增大鼠MSCs,经流式细胞仪检测其细胞周期及表面标志后,用DAPI标记,经尾静脉注入胰腺损伤模型大鼠体内,15d后,在激光共聚焦显微镜下观察MSCs在大鼠胰腺组织的定位,组织病理切片观察胰腺损伤组织的病理改变,PCR检测胰腺损伤区组织的Sry基因。结果体外纯化、扩增、富集的MSCs经流式细胞仪检测,86.67%的细胞处于G0/G1期,细胞表面CD34呈阴性表达,CD44呈阳性表达。DAPI标记的MSCs移植治疗15d后,激光共聚焦显微镜下可见,MSCs在损伤的胰腺组织中多见,在正常胰腺组织中偶见;组织病理切片可见损伤的胰腺组织结构开始恢复,胰岛再建;PCR结果显示,治疗组胰腺组织可扩增出Sry基因。结论MSCs对大鼠胰腺损伤组织可能具有修复作用。     

MSCs Become Collagen-Type I Producing Cells with Different Phenotype in Allogeneic and Syngeneic Bone Marrow Transplantation

Mesenchymal stem cells (MSCs) have been widely used in therapeutic applications for many decades. However, more and more evidence suggests that factors such as the site of origin and pre-implantation treatment have a crucial impact on the result. This study investigates the role of freshly isolated MSCs in the lacrimal gland after allogeneic transplantation. For this purpose, MSCs from transgenic GFP mice were isolated and transplanted into allogeneic and syngeneic recipients. While the syngeneic MSCs maintained a spherical shape, allogeneic MSCs engrafted into the tissue as spindle-shaped cells in the interstitial stroma. Furthermore, the MSCs produced collagen type I in more than 85% to 95% of the detected GFP⁺ MSCs in the recipients of both models, supposedly contributing to pathogenic fibrosis in allogeneic recipients compared to syngeneic models. These findings indicate that allogeneic MSCs act completely differently from syngeneic MSCs, highlighting the importance of understanding the exact mechanisms behind MSCs.     

骨髓间充质干细胞在脑缺血模型大鼠脑组织中的迁徙、定居及组织修复作用

目的观察骨髓间充质干细胞(MSCs)移植后,在缺血性脑损伤大鼠脑组织中的迁徙、定居及组织修复作用。方法体外分离、纯化及培养MSCs。线栓法制备Wester大鼠脑缺血模型,经颈动脉移植DAPI标记的MSCs,通过激光共聚焦显微镜,分别在24h、5d及10d观察MSCs在脑组织内的迁徙及定居。采用三苯四氮唑活组织脑片染色方法,观察MSCs治疗对脑组织损伤的修复作用。结果MSCs经颈动脉移植后,在24h即出现在脑损伤区域血管内,第5天在血管周围组织内开始弥散,第10天在损伤区域可见广泛弥散。MSCs治疗20d后,脑片染色显示坏死区域减少。结论动脉移植MSCs后,MSCs首先出现在大鼠缺血性脑损伤区域血管内,然后在周围组织弥散,并可能参与损伤区血管及组织的修复。     

The Cross-Talk between Mesenchymal Stem Cells and Immune Cells in Tissue Repair and Regeneration

Mesenchymal stem cells (MSCs) are self-renewable, rapidly proliferating, multipotent stem cells which reside in almost all post-natal tissues. MSCs possess potent immunoregulatory properties and, in juxtacrine and paracrine manner, modulate phenotype and function of all immune cells that participate in tissue repair and regeneration. Additionally, MSCs produce various pro-angiogenic factors and promote neo-vascularization in healing tissues, contributing to their enhanced repair and regeneration. In this review article, we summarized current knowledge about molecular mechanisms that regulate the crosstalk between MSCs and immune cells in tissue repair and regeneration.     

Protective Effects of a Hyaluronan-Binding Peptide (P15-1) on Mesenchymal Stem Cells in an Inflammatory Environment

Mesenchymal stem cells (MSCs) obtained from various sources, including bone marrow, have been proposed as a therapeutic strategy for the improvement of tissue repair/regeneration, including the repair of cartilage defects or lesions. Often the highly inflammatory environment after injury or during diseases, however, greatly diminishes the therapeutic and reparative effectiveness of MSCs. Therefore, the identification of novel factors that can protect MSCs against an inflammatory environment may enhance the effectiveness of these cells in repairing tissues, such as articular cartilage. In this study, we investigated whether a peptide (P15-1) that binds to hyaluronan (HA), a major component of the extracellular matrix of cartilage, protects bone-marrow-derived MSCs (BMSCs) in an inflammatory environment. The results showed that P15-1 reduced the mRNA levels of catabolic and inflammatory markers in interleukin-1beta (IL-1β)-treated human BMSCs. In addition, P15-1 enhanced the attachment of BMSCs to HA-coated tissue culture dishes and stimulated the chondrogenic differentiation of the multipotential murine C3H/10T1/2 MSC line in a micromass culture. In conclusion, our findings suggest that P15-1 may increase the capacity of BMSCs to repair cartilage via the protection of these cells in an inflammatory environment and the stimulation of their attachment to an HA-containing matrix and chondrogenic differentiation.     

Pulsed Electromagnetic Field Stimulation in Osteogenesis and Chondrogenesis: Signaling Pathways and Therapeutic Implications

Mesenchymal stem cells (MSCs) are the main cell players in tissue repair and thanks to their self-renewal and multi-lineage differentiation capabilities, they gained significant attention as cell source for tissue engineering (TE) approaches aimed at restoring bone and cartilage defects. Despite significant progress, their therapeutic application remains debated: the TE construct often fails to completely restore the biomechanical properties of the native tissue, leading to poor clinical outcomes in the long term. Pulsed electromagnetic fields (PEMFs) are currently used as a safe and non-invasive treatment to enhance bone healing and to provide joint protection. PEMFs enhance both osteogenic and chondrogenic differentiation of MSCs. Here, we provide extensive review of the signaling pathways modulated by PEMFs during MSCs osteogenic and chondrogenic differentiation. Particular attention has been given to the PEMF-mediated activation of the adenosine signaling and their regulation of the inflammatory response as key player in TE approaches. Overall, the application of PEMFs in tissue repair is foreseen: (1) in vitro: to improve the functional and mechanical properties of the engineered construct; (2) in vivo: (i) to favor graft integration, (ii) to control the local inflammatory response, and (iii) to foster tissue repair from both implanted and resident MSCs cells.     

Evaluating the Effect of Hypoxia on Human Adult Mesenchymal Stromal Cell Chondrogenesis In Vitro: A Systematic Review

Human adult mesenchymal stromal cells (MSCs) from a variety of sources may be used to repair defects in articular cartilage by inducing them into chondrogenic differentiation. The conditions in which optimal chondrogenic differentiation takes place are an area of interest in the field of tissue engineering. Chondrocytes exist in vivo in a normally hypoxic environment and thus it has been suggested that exposing MSCs to hypoxia may also contribute to a beneficial effect on their differentiation. There are two main stages in which MSCs can be exposed to hypoxia, the expansion phase when cells are cultured, and the differentiation phase when cells are induced with a chondrogenic medium. This systematic review sought to explore the effect of hypoxia at these two stages on human adult MSC chondrogenesis in vitro. A literature search was performed on PubMed, EMBASE, Medline via Ovid, and Cochrane, and 24 studies were ultimately included. The majority of these studies showed that hypoxia during the expansion phase or the differentiation phase enhances at least some markers of chondrogenic differentiation in adult MSCs. These results were not always demonstrated at the protein level and there were also conflicting reports. Studies evaluating continuous exposure to hypoxia during the expansion and differentiation phases also had mixed results. These inconsistent results can be explained by the heterogeneity of studies, including factors such as different sources of MSCs used, donor variability, level of hypoxia used in each study, time exposed to hypoxia, and differences in culture methodology.     

Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells and Their Contribution to Angiogenic Processes in Tissue Regeneration

Mesenchymal stem/stromal cells (MSCs) are widely described in the context of their regenerative and immunomodulatory activity. MSCs are isolated from various tissues and organs. The most frequently described sources are bone marrow and adipose tissue. As stem cells, MSCs are able to differentiate into other cell lineages, but they are usually reported with respect to their paracrine potential. In this review, we focus on MSCs derived from adipose tissue (AT-MSCs) and their secretome in regeneration processes. Special attention is given to the contribution of AT-MSCs and their derivatives to angiogenic processes described mainly in the context of angiogenic dysfunction. Finally, we present clinical trials registered to date that concern the application of AT-MSCs and their secretome in various medical conditions.     

Immunomodulatory Mechanisms of Mesenchymal Stem Cells and Their Potential Clinical Applications

Mesenchymal stem cells (MSCs) are multipotent stem cells with the capacity of self-renewal, homing, and low immunogenicity. These distinct biological characteristics have already shown immense potential in regenerative medicine. MSCs also possess immunomodulatory properties that can maintain immune homeostasis when the immune response is over-activated or under-activated. The secretome of MSCs consists of cytokines, chemokines, signaling molecules, and growth factors, which effectively contribute to the regulation of immune and inflammatory responses. The immunomodulatory effects of MSCs can also be achieved through direct cell contact with microenvironmental factors and immune cells. Furthermore, preconditioned and engineered MSCs can specifically improve the immunomodulation effects in diverse clinical applications. These multifunctional properties of MSCs enable them to be used as a prospective therapeutic strategy to treat immune disorders, including autoimmune diseases and incurable inflammatory diseases. Here we review the recent exploration of immunomodulatory mechanisms of MSCs and briefly discuss the promotion of the genetically engineered MSCs. Additionally, we review the potential clinical applications of MSC-mediated immunomodulation in four types of immune diseases, including systemic lupus erythematosus, Crohn’s disease, graft-versus-host disease, and COVID-19.     

The Regulatory Role of Signaling Crosstalk in Hypertrophy of MSCs and Human Articular Chondrocytes

Hypertrophic differentiation of chondrocytes is a main barrier in application of mesenchymal stem cells (MSCs) for cartilage repair. In addition, hypertrophy occurs occasionally in osteoarthritis (OA). Here we provide a comprehensive review on recent literature describing signal pathways in the hypertrophy of MSCs-derived in vitro differentiated chondrocytes and chondrocytes, with an emphasis on the crosstalk between these pathways. Insight into the exact regulation of hypertrophy by the signaling network is necessary for the efficient application of MSCs for articular cartilage repair and for developing novel strategies for curing OA. We focus on articles describing the role of the main signaling pathways in regulating chondrocyte hypertrophy-like changes. Most studies report hypertrophic differentiation in chondrogenesis of MSCs, in both human OA and experimental OA. Chondrocyte hypertrophy is not under the strict control of a single pathway but appears to be regulated by an intricately regulated network of multiple signaling pathways, such as WNT, Bone morphogenetic protein (BMP)/Transforming growth factor-β (TGFβ), Parathyroid hormone-related peptide (PTHrP), Indian hedgehog (IHH), Fibroblast growth factor (FGF), Insulin like growth factor (IGF) and Hypoxia-inducible factor (HIF). This comprehensive review describes how this intricate signaling network influences tissue-engineering applications of MSCs in articular cartilage (AC) repair, and improves understanding of the disease stages and cellular responses within an OA articular joint.     

The Use of Infrapatellar Fat Pad-Derived Mesenchymal Stem Cells in Articular Cartilage Regeneration: A Review

Cartilage is frequently damaged with a limited capacity for repair. Current treatment strategies are insufficient as they form fibrocartilage as opposed to hyaline cartilage, and do not prevent the progression of degenerative changes. There is increasing interest in the use of autologous mesenchymal stem cells (MSC) for tissue regeneration. MSCs that are used to treat articular cartilage defects must not only present a robust cartilaginous production capacity, but they also must not cause morbidity at the harvest site. In addition, they should be easy to isolate from the tissue and expand in culture without terminal differentiation. The source of MSCs is one of the most important factors that may affect treatment. The infrapatellar fat pad (IPFP) acts as an important reservoir for MSC and is located in the anterior compartment of the knee joint in the extra-synovial area. The IPFP is a rich source of MSCs, and in this review, we discuss studies that demonstrate that these cells have shown many advantages over other tissues in terms of ease of isolation, expansion, and chondrogenic differentiation. Future studies in articular cartilage repair strategies and suitable extraction as well as cell culture methods will extend the therapeutical application of IPFP-derived MSCs into additional orthopedic fields, such as osteoarthritis. This review provides the latest research concerning the use of IPFP-derived MSCs in the treatment of articular cartilage damage, providing critical information for the field to grow.     

Therapeutic Potential of Differentiated Mesenchymal Stem Cells for Treatment of Osteoarthritis

Osteoarthritis (OA) is a chronic, progressive, and irreversible degenerative joint disease. Conventional OA treatments often result in complications such as pain and limited activity. However, transplantation of mesenchymal stem cells (MSCs) has several beneficial effects such as paracrine effects, anti-inflammatory activity, and immunomodulatory capacity. In addition, MSCs can be differentiated into several cell types, including chondrocytes, osteocytes, endothelia, and adipocytes. Thus, transplantation of MSCs is a suggested therapeutic tool for treatment of OA. However, transplanted naïve MSCs can cause problems such as heterogeneous populations including differentiated MSCs and undifferentiated cells. To overcome this problem, new strategies for inducing differentiation of MSCs are needed. One possibility is the application of microRNA (miRNA) and small molecules, which regulate multiple molecular pathways and cellular processes such as differentiation. Here, we provide insight into possible strategies for cartilage regeneration by transplantation of differentiated MSCs to treat OA patients.     

Mesenchymal Stem Cell-Derived Microparticles: A Promising Therapeutic Strategy

Mesenchymal stem cells (MSCs) are multipotent stem cells that give rise to various cell types of the mesodermal germ layer. Because of their unique ability to home in on injured and cancerous tissues, MSCs are of great potential in regenerative medicine. MSCs also contribute to reparative processes in different pathological conditions, including cardiovascular diseases and cancer. However, many studies have shown that only a small proportion of transplanted MSCs can actually survive and be incorporated into host tissues. The effects of MSCs cannot be fully explained by their number. Recent discoveries suggest that microparticles (MPs) derived from MSCs may be important for the physiological functions of their parent. Though the physiological role of MSC-MPs is currently not well understood, inspiring results indicate that, in tissue repair and anti-cancer therapy, MSC-MPs have similar pro-regenerative and protective properties as their cellular counterparts. Thus, MSC-MPs represent a promising approach that may overcome the obstacles and risks associated with the use of native or engineered MSCs.     

Metabolic Reconfiguration Activates Stemness and Immunomodulation of PDLSCs

Periodontal ligament derived stem cells (PDLSC) are adult multipotent mesenchymal-like stem cells (MSCs) that can induce a promising immunomodulation to interact with immune cells for disease treatment. Metabolic reconfiguration has been shown to be involved in the immunomodulatory activity of MSCs. However, the underlying mechanisms are largely unknown, and it remains a challenging to establish a therapeutic avenue to enhance immunomodulation of endogenous stem cells for disease management. In the present study, RNA-sequencing (RNA-seq) analysis explores that curcumin significantly promotes PDLSC function through activation of MSC-related markers and metabolic pathways. In vitro stem cell characterization further confirms that self-renewal and multipotent differentiation capabilities are largely elevated in curcumin treated PDLSCs. Mechanistically, RNA-seq reveals that curcumin activates ERK and mTOR cascades through upregulating growth factor pathways for metabolic reconfiguration toward glycolysis. Interestingly, PDLSCs immunomodulation is significantly increased after curcumin treatment through activation of prostaglandin E2-Indoleamine 2,3 dioxygenase (PGE2-IDO) signaling, whereas inhibition of glycolysis activity by 2-deoxyglucose (2-DG) largely blocked immunomodulatory capacity of PDLSCs. Taken together, this study provides a novel pharmacological approach to activate endogenous stem cells through metabolic reprogramming for immunomodulation and tissue regeneration.     

骨髓间充质干细胞对电离辐射诱发的小鼠胸腺瘤的抑制作用

目的观察骨髓间充质干细胞(MSCs)对电离辐射诱发的小鼠胸腺瘤的抑制作用。方法采用经典Kaplan法复制电离辐射诱发的小鼠胸腺瘤模型。应用全骨髓贴壁法分离培养C57BL/6小鼠MSCs,DAPI标记,经尾静脉注入荷瘤小鼠后,分别于1、5、10d处死小鼠,取胸腺组织,激光共聚焦显微镜下观察MSCs在胸腺瘤组织中的定位;第1次全身大剂量照射后6个月取胸腺组织,HE染色观察胸腺组织的病理变化,并判断成瘤情况。结果激光共聚焦显微镜下观察可见,MSCs经尾静脉输注后可迁徙至小鼠胸腺组织内;病理观察显示,胸腺组织皮髓质结构清楚,淋巴样肿瘤细胞较少,细胞形态、大小不一,偶见核分裂象;MSCs输注使辐射诱导的胸腺瘤成瘤率由57.00%±9.78%降低至37.50%±7.55%。结论已成功建立辐射诱发的小鼠胸腺瘤模型;输注的MSCs可迁徙至胸腺组织中,并降低胸腺瘤的成瘤率。     

bFGF促增殖后骨髓间充质干细胞向多巴胺能神经元样细胞的分化

目的探讨碱性成纤维细胞生长因子(Basic fibroblast growth factor,bFGF)体外作用于骨髓间充质干细胞(Mesen-chymal stem cells,MSCs)后,诱导其向神经元样细胞和多巴胺能神经元样细胞定向分化的情况。方法从鼠骨髓中获得MSCs,培养传代,用MTT法检测bFGF对骨髓MSCs生长的影响。10 ng/ml bFGF作用2 d后,通过IBMX、细胞因子bFGF、GDNFI、L-1β、中脑神经胶质细胞条件培养基和中脑神经细胞膜碎片等分组联合诱导骨髓MSCs向神经元样细胞、多巴胺能神经元样细胞分化,免疫细胞化学方法鉴定特异标志NSE、MAP-2a,b和TH的表达。结果在一定范围内,bFGF对骨髓MSCs具有明显的促增殖作用。分化的神经元样细胞表达NSE、MAP-2a,b和TH,联合应用GDNFI、L-1β、中脑条件培养基和中脑神经细胞膜碎片诱导7 d后,NSE阳性率为(27.774±2.747)%,MAP-2a,b为(28.006±3.080)%,TH为(3.098±0.352)%。结论体外骨髓MSCs被诱导分化成神经元样细胞和多巴胺能神经元样细胞,为帕金森等中枢神经系统疾病的细胞移植治疗奠定了基础。     

Biophysical Modulation of Mesenchymal Stem Cell Differentiation in the Context of Skeletal Repair

A prominent feature of the skeleton is its ability to remodel in response to biophysical stimuli and to repair under varied biophysical conditions. This allows the skeleton considerable adaptation to meet its physiological roles of stability and movement. Skeletal cells and their mesenchymal precursors exist in a native environment rich with biophysical signals, and they sense and respond to those signals to meet organismal demands of the skeleton. While mechanical strain is the most recognized of the skeletal biophysical stimuli, signaling phenomena also include fluid flow, hydrostatic pressure, shear stress, and ion-movement-related electrokinetic phenomena including, prominently, streaming potentials. Because of the complex interactions of these electromechanical signals, it is difficult to isolate the significance of each. The application of external electrical and electromagnetic fields allows an exploration of the effects of these stimuli on cell differentiation and extra-cellular matrix formation in the absence of mechanical strain. This review takes a distinctly translational approach to mechanistic and preclinical studies of differentiation and skeletal lineage commitment of mesenchymal cells under biophysical stimulation. In vitro studies facilitate the examination of isolated cellular responses while in vivo studies permit the observation of cell differentiation and extracellular matrix synthesis.     

大鼠骨髓间充质干细胞的体外分离培养与鉴定

目的建立大鼠骨髓间充质干细胞(Mesenchymal stem cells,MSCs)体外分离培养及鉴定的方法 ,为MSCs的系列研究奠定基础。方法采用全骨髓直接贴壁筛选法分离培养MSCs并传代,倒置相差显微镜下观察细胞形态,以MTT法检测细胞增殖水平并绘制生长曲线。取第3代MSCs,流式细胞术检测细胞周期和细胞表型,应用成骨细胞诱导液和脂肪样细胞诱导液诱导MSCs定向分化,鉴定其分化能力。结果全骨髓细胞培养5d,镜下可见贴壁细胞增殖明显,细胞形态较均一,大部分呈梭形,7d左右可传代,经2～3次传代后细胞呈单一梭形的成纤维样细胞,即MSCs;细胞生长曲线呈S形;经流式细胞仪检测,MSCs细胞76.01%处于G0/G1期,7.13%处于G2/M期,16.86%处于S期;MSCs表面不表达CD34;在特定诱导液作用下,MSCs可分别向成骨样细胞及脂肪样细胞分化。结论已成功建立了分离培养及鉴定MSCs的方法 ,可用来评价体外培养的MSCs。