Biohybrid Bovine Bone Matrix for Controlled Release of Mesenchymal Stem/Stromal Cell Lyosecretome: A Device for Bone Regeneration

SmartBone^® (SB) is a biohybrid bone substitute advantageously proposed as a class III medical device for bone regeneration in reconstructive surgeries (oral, maxillofacial, orthopedic, and oncology). In the present study, a new strategy to improve SB osteoinductivity was developed. SB scaffolds were loaded with lyosecretome, a freeze-dried formulation of mesenchymal stem cell (MSC)-secretome, containing proteins and extracellular vesicles (EVs). Lyosecretome-loaded SB scaffolds (SBlyo) were prepared using an absorption method. A burst release of proteins and EVs (38% and 50% after 30 min, respectively) was observed, and then proteins were released more slowly with respect to EVs, most likely because they more strongly adsorbed onto the SB surface. In vitro tests were conducted using adipose tissue-derived stromal vascular fraction (SVF) plated on SB or SBlyo. After 14 days, significant cell proliferation improvement was observed on SBlyo with respect to SB, where cells filled the cavities between the native trabeculae. On SB, on the other hand, the process was still present, but tissue formation was less organized at 60 days. On both scaffolds, cells differentiated into osteoblasts and were able to mineralize after 60 days. Nonetheless, SBlyo showed a higher expression of osteoblast markers and a higher quantity of newly formed trabeculae than SB alone. The quantification analysis of the newly formed mineralized tissue and the immunohistochemical studies demonstrated that SBlyo induces bone formation more effectively. This osteoinductive effect is likely due to the osteogenic factors present in the lyosecretome, such as fibronectin, alpha-2-macroglobulin, apolipoprotein A, and TGF-β.     

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.     

Mechanotransduction in Mesenchymal Stem Cells (MSCs) Differentiation: A Review

Mechanotransduction is the process by which physical force is converted into a biochemical signal that is used in development and physiology; meanwhile, it is intended for the ability of cells to sense and respond to mechanical forces by activating intracellular signals transduction pathways and the relative phenotypic adaptation. It encompasses the role of mechanical stimuli for developmental, morphological characteristics, and biological processes in different organs; the response of cells to mechanically induced force is now also emerging as a major determinant of disease. Due to fluid shear stress caused by blood flowing tangentially across the lumen surface, cells of the cardiovascular system are typically exposed to a variety of mechanotransduction. In the body, tissues are continuously exposed to physical forces ranging from compression to strain, which is caused by fluid pressure and compressive forces. Only lately, though, has the importance of how forces shape stem cell differentiation into lineage-committed cells and how mechanical forces can cause or exacerbate disease besides organizing cells into tissues been acknowledged. Mesenchymal stem cells (MSCs) are potent mediators of cardiac repair which can secret a large array of soluble factors that have been shown to play a huge role in tissue repair. Differentiation of MSCs is required to regulate mechanical factors such as fluid shear stress, mechanical strain, and the rigidity of the extracellular matrix through various signaling pathways for their use in regenerative medicine. In the present review, we highlighted mechanical influences on the differentiation of MSCs and the general factors involved in MSCs differentiation. The purpose of this study is to demonstrate the progress that has been achieved in understanding how MSCs perceive and react to their mechanical environment, as well as to highlight areas where more research has been performed in previous studies to fill in the gaps.     

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.     

Exosome Degeneration in Mesenchymal Stem Cells Derived from Patients with Type 1 Diabetes Mellitus

Type 1 diabetes mellitus is characterized by the destruction of pancreatic β-cells and requires the regeneration of these destroyed pancreatic β-cells for radical treatment. The degeneration of organelles in stem cells compromises stem cell quality; however, organelles in the mesenchymal stem cells of patients with type 1 diabetes mellitus have not been characterized previously. In this study, we use transmission electron microscopy to evaluate the degeneration of organelles in adipose-derived stem cells of patients with type 1 diabetes mellitus (T1DM ADSCs). Compared to adipose-derived stem cells from healthy humans, T1DM ADSCs degenerate differently, characterized by prominent enlarged spherical vesicles. The exosomes of T1DM ADSCs are found to be enlarged, reduced in number, and increased in the percentage of those positive for tetraspanin CD9. The findings of this study provide insight into the characteristics of stem cells in patients with type 1 diabetes mellitus.     

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.     

Secretome from Human Mesenchymal Stem Cells-Derived Endothelial Cells Promotes Wound Healing in a Type-2 Diabetes Mouse Model

Tissue regeneration is often impaired in patients with metabolic disorders such as diabetes mellitus and obesity, exhibiting reduced wound repair and limited regeneration capacity. We and others have demonstrated that wound healing under normal metabolic conditions is potentiated by the secretome of human endothelial cell-differentiated mesenchymal stem cells (hMSC-EC). However, it is unknown whether this effect is sustained under hyperglycemic conditions. In this study, the wound healing effect of secretomes from undifferentiated human mesenchymal stem cells (hMSC) and hMSC-EC in a type-2 diabetes mouse model was analyzed. hMSC were isolated from human Wharton’s jelly and differentiated into hMSC-EC. hMSC and hMSC-EC secretomes were analyzed and their wound healing capacity in C57Bl/6J mice fed with control (CD) or high fat diet (HFD) was evaluated. Our results showed that hMSC-EC secretome enhanced endothelial cell proliferation and wound healing in vivo when compared with hMSC secretome. Five soluble proteins (angiopoietin-1, angiopoietin-2, Factor de crecimiento fibroblástico, Matrix metallopeptidase 9, and Vascular Endothelial Growth Factor) were enriched in hMSC-EC secretome in comparison to hMSC secretome. Thus, the five recombinant proteins were mixed, and their pro-healing property was evaluated in vitro and in vivo. Functional analysis demonstrated that a cocktail of these proteins enhanced the wound healing process similar to hMSC-EC secretome in HFD mice. Overall, our results show that hMSC-EC secretome or a combination of specific proteins enriched in the hMSC-EC secretome enhanced wound healing process under hyperglycemic conditions.     

Addition of Adipose-Derived Stem Cells to Mesenchymal Stem Cell Sheets Improves Bone Formation at an Ectopic Site

To determine the effect of adipose-derived stem cells (ADSCs) added to bone marrow-derived mesenchymal stem cell (MSC) sheets on bone formation at an ectopic site. We isolated MSCs and ADSCs from the same rabbits. We then prepared MSC sheets for implantation with or without ADSCs subcutaneously in the backs of severe combined immunodeficiency (SCID) mice. We assessed bone formation at eight weeks after implantation by micro-computed tomography and histological analysis. In osteogenic medium, MSCs grew to form multilayer sheets containing many calcium nodules. MSC sheets without ADSCs formed bone-like tissue; although neo-bone and cartilage-like tissues were sparse and unevenly distributed by eight weeks after implantation. In comparison, MSC sheets with ADSCs promoted better bone regeneration as evidenced by the greater density of bone, increased mineral deposition, obvious formation of blood vessels, large number of interconnected ossified trabeculae and woven bone structures, and greater bone volume/total volume within the composite constructs. Our results indicate that although sheets of only MSCs have the potential to form tissue engineered bone at an ectopic site, the addition of ADSCs can significantly increase the osteogenic potential of MSC sheets. Thus, the combination of MSC sheets with ADSCs may be regarded as a promising therapeutic strategy to stimulate bone regeneration.     

A Single-Cell Raman Spectroscopy Analysis of Bone Marrow Mesenchymal Stem/Stromal Cells to Identify Inter-Individual Diversity

The heterogeneity of stem cells represents the main challenge in regenerative medicine development. This issue is particularly pronounced when it comes to the use of primary mesenchymal stem/stromal cells (MSCs) due to a lack of identification markers. Considering the need for additional approaches in MSCs characterization, we applied Raman spectroscopy to investigate inter-individual differences between bone marrow MSCs (BM-MSCs). Based on standard biological tests, BM-MSCs of analyzed donors fulfill all conditions for their characterization, while no donor-related specifics were observed in terms of BM-MSCs morphology, phenotype, multilineage differentiation potential, colony-forming capacity, expression of pluripotency-associated markers or proliferative capacity. However, examination of BM-MSCs at a single-cell level by Raman spectroscopy revealed that despite similar biochemical background, fine differences in the Raman spectra of BM-MSCs of each donor can be detected. After extensive principal component analysis (PCA) of Raman spectra, our study revealed the possibility of this method to diversify BM-MSCs populations, whereby the grouping of cell populations was most prominent when cell populations were analyzed in pairs. These results indicate that Raman spectroscopy, as a label-free assay, could have a huge potential in understanding stem cell heterogeneity and sorting cell populations with a similar biochemical background that can be significant for the development of personalized therapy approaches.     

Regenerative Potential of Mesenchymal Stem Cells’ (MSCs) Secretome for Liver Fibrosis Therapies

Chronic liver injuries lead to liver fibrosis and then to end-stage liver cirrhosis. Liver transplantation is often needed as a course of treatment for patients in critical conditions, but limitations associated with transplantation prompted the continuous search for alternative therapeutic strategies. Cell therapy with stem cells has emerged as an attractive option in order to stimulate tissue regeneration and liver repair. Transplanted mesenchymal stem cells (MSCs) could trans-differentiate into hepatocyte-like cells and, moreover, show anti-fibrotic and immunomodulatory effects. However, cell transplantation may lead to some uncontrolled side effects, risks associated with tumorigenesis, and cell rejection. MSCs’ secretome includes a large number of soluble factors and extracellular vesicles (EVs), through which they exert their therapeutic role. This could represent a cell-free strategy, which is safer and more effective than MSC transplantation. In this review, we focus on cell therapies based on MSCs and how the MSCs’ secretome impacts the mechanisms associated with liver diseases. Moreover, we discuss the important therapeutic role of EVs and how their properties could be further used in liver regeneration.     

Secretome of Mesenchymal Stem Cells from Consecutive Hypoxic Cultures Promotes Resolution of Lung Inflammation by Reprogramming Anti-Inflammatory Macrophages

The secretome from hypoxia-preconditioned mesenchymal stem cells (MSCs) has been shown to promote resolution of inflammation and alleviate acute lung injury (ALI) through its immunomodulatory function. However, the effects of consecutive hypoxic culture on immunomodulatory function of the MSCs secretome are largely unclarified. Here, we intend to investigate the effects of consecutive hypoxia on therapeutic efficacy of conditioned medium derived from MSCs (MSCs-CM) in alleviating ALI. Human umbilical cord-derived MSCs (UC-MSCs) were consecutively cultured in 21% O₂ (Nor-MSCs) or in 1% O₂ (Hypo-MSCs) from passage 0. Their conditioned medium (Nor-CM and Hypo-CM respectively) was collected and administered into ALI models. Our findings confirmed that Hypo-MSCs exhibited increased proliferation ability and decreased cell senescence compared with Nor-MSCs. Consecutive hypoxia promoted UC-MSCs to secrete immunomodulatory cytokines, such as insulin-like growth factor 1(IGF1), IL10, TNFα-stimulated gene 6(TSG6), TGFβ, and prostaglandin E2 (PGE2). Both Nor-CM and Hypo-CM could effectively limit lung inflammation, promote efferocytosis and modulate anti-inflammatory polarization of lung macrophages in ALI models. Moreover, the effects of Hypo-CM were more potent than Nor-CM. Taken together, our findings indicate that consecutive hypoxic cultures could not only promote both proliferation and quality of UC-MSCs, but also enhance the therapeutic efficacy of their secretome in mitigating lung inflammation by promoting efferocytosis and anti-inflammatory polarization of macrophages.     

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。     

Tissue-Protective and Anti-Inflammatory Landmark of PRP-Treated Mesenchymal Stromal Cells Secretome for Osteoarthritis

Bone-marrow-mesenchymal-stromal-cells (BMSCs)- and platelet-rich-plasma (PRP)-based therapies have shown potential for treating osteoarthritis (OA). Recently, the combination of these two approaches was proposed, with results that overcame those observed with the separate treatments, indicating a possible role of PRP in ameliorating BMSCs’ regenerative properties. Since a molecular fingerprint of BMSCs cultivated in the presence of PRP is missing, the aim of this study was to characterize the secretome in terms of soluble factors and extracellular-vesicle (EV)-embedded miRNAs from the perspective of tissues, pathways, and molecules which frame OA pathology. One hundred and five soluble factors and one hundred eighty-four EV-miRNAs were identified in the PRP-treated BMSCs’ secretome, respectively. Several soluble factors were related to the migration of OA-related immune cells, suggesting the capacity of BMSCs to attract lympho-, mono-, and granulocytes and modulate their inflammatory status. Accordingly, several EV-miRNAs had an immunomodulating role at both the single-factor and cell level, together with the ability to target OA-characterizing extracellular-matrix-degrading enzymes and cartilage destruction pathways. Overall, anti-inflammatory and protective signals far exceeded inflammation and destruction cues for cartilage, macrophages, and T cells. This study demonstrates that BMSCs cultivated in the presence of PRP release therapeutic molecules and give molecular ground for the use of this combined and innovative therapy for OA treatment.     

Role of Mesenchymal Stem/Stromal Cells in Coagulation

Mesenchymal stem/stromal cells (MSCs) are widely used in disease models in order to control several phases in the response to injuries, immune reaction, wound healing, and regeneration. MSCs can act upon both the innate and adaptive immune systems and target a broad number of functions, such as the secretion of cytokines, proteolytic enzymes, angiogenic factors, and the regulating of cell proliferation and survival. The role of MSCs in coagulation has been less studied. This review evaluates the properties and main functions of MSCs in coagulation. MSCs can regulate coagulation in a wide range of pathways. MSCs express and release tissue factors (TF), one of the key regulators of the extrinsic coagulation pathways; MSCs can trigger platelet production and contribute to platelet activation. Altogether, MSCs seem to have a pro-thrombotic role and their superior characterization prior to their administration is necessary in order to prevent adverse coagulation events.     

Characterization of Extracellular Vesicles from Preconditioned Human Adipose-Derived Stromal/Stem Cells

Cell-free therapy using extracellular vesicles (EVs) from adipose-derived mesenchymal stromal/stem cells (ASCs) seems to be a safe and effective therapeutic option to support tissue and organ regeneration. The application of EVs requires particles with a maximum regenerative capability and hypoxic culture conditions as an in vitro preconditioning regimen has been shown to alter the molecular composition of released EVs. Nevertheless, the EV cargo after hypoxic preconditioning has not yet been comprehensively examined. The aim of the present study was the characterization of EVs from hypoxic preconditioned ASCs. We investigated the EV proteome and their effects on renal tubular epithelial cells in vitro. While no effect of hypoxia was observed on the number of released EVs and their protein content, the cargo of the proteins was altered. Proteomic analysis showed 41 increased or decreased proteins, 11 in a statistically significant manner. Furthermore, the uptake of EVs in epithelial cells and a positive effect on oxidative stress in vitro were observed. In conclusion, culture of ASCs under hypoxic conditions was demonstrated to be a promising in vitro preconditioning regimen, which alters the protein cargo and increases the anti-oxidative potential of EVs. These properties may provide new potential therapeutic options for regenerative medicine.     

骨髓间充质干细胞对海藻酸钙胶珠内神经干细胞增殖与分化的作用

神经干细胞(neural stem cells,NSCs)移植治疗神经损伤被认为是具有潜在应用价值的手段,但其来源困难;骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)以其所具有的诸多优点,为神经损伤的治疗提供了一个新的思路。而BMSCs是否是通过作用于内源性的NSCs来促进神经修复,仍存在着争议。今采用海藻酸钙胶珠将NSCs包囊培养至一定大小的神经球后,再与BMSCs进行共培养,考察BMSCs对生长在海藻酸钙胶珠内的NSCs增殖与分化的作用,探讨BMSCs移植治疗神经疾病与损伤的作用机理。共培养过程中观察神经球结构的变化;共培养结束后计算NSCs的增殖倍数,对增殖条件下共培养的NSCs表型和多向分化潜能进行免疫荧光染色鉴定;对分化条件下共培养的NSCs向不同神经细胞分化的能力进行流式细胞仪检测。结果表明,BMSCs可使生长于支架内的NSCs迁出细胞球,对NSCs的增殖没有明显影响;但能够明显影响NSCs的分化,使其向少突胶质细胞分化的能力增加3倍,向星形胶质细胞分化的能力减弱1倍,而向神经元细胞分化的能力没有明显变化。BMSCs有可能是通过分泌某些因子增加了NSCs迁移及向少突胶质细胞分化的能力,从而促进神经损伤的修复。     

Hematopoietic–Mesenchymal Signals Regulate the Properties of Mesenchymal Stem Cells

It is well known that the properties of hematopoietic stem/progenitor cells (HSCs), such as their self-renewal ability and multipotency, are maintained through interactions with mesenchymal stem/stromal cells (MSCs). MSCs are rare cells that are present in the bone marrow and are useful for clinical applications due to their functional ability. To obtain the necessary number of cells, MSCs must be cultured to expand, but this causes a remarkable decrease in stem cell properties, such as multipotency and proliferation ability. In this study, we show that the c-Mpl signal, which is related to the maintenance of hematopoietic stem cells, has an important effect on the proliferation and differentiation ability of MSCs. Utilizing a co-culture system comprising MSCs and HSCs, it is suggested that signaling from hematopoietic cells to MSCs supports cell proliferation. Interestingly, the enhanced proliferation ability of the HSCs was decreased in c-Mpl knock-out HSCs (c-Mpl-KO). In addition, the MSCs co-cultured with c-Mpl-KO HSCs had reduced MSC marker expression (PDGFRa and Sca-1) compared to the MSCs co-cultured with c-Mpl-wild-type HSCs. These results suggest that a hematopoietic–mesenchymal signal exists, and that the state of the HSCs is important for the stability of MSC properties.     

骨髓间充质干细胞对胰腺损伤模型大鼠血清生化指标的影响

目的观察大鼠骨髓间充质干细胞(MSCs)对胰腺损伤模型大鼠血清生化指标的影响。方法胰腺结扎建立大鼠胰腺损伤模型;体外分离纯化及培养大鼠的MSCs,流式细胞术检测细胞周期及细胞表面标志,经尾静脉回输治疗胰腺损伤,逐日观察,并于回输后24h、48h、7d和15d,分别采血,分离血清,检测血淀粉酶与脂肪酶含量。结果分离纯化的MSCs细胞周期中,86.67%处于G0/G1期,表达MSCs表面标志CD44,不表达造血干细胞表面标志CD34。治疗组在回输MSCs15d后,肉眼可见坏死的胰腺组织外观基本恢复正常,24h、48h和7d,血清淀粉酶含量均低于模型组,且差异有统计学意义,在15d时接近于模型组,差异无统计学意义;在24h、48h、7d和15d时,治疗组血清中脂肪酶含量均低于模型组,且差异有统计学意义;24h、48h、7d和15d,治疗组血清中血淀粉酶和脂肪酶含量仍高于正常对照组,且差异有统计学意义。结论骨髓间充质干细胞对胰腺组织损伤的模型大鼠具有治疗作用。