Human Bone Marrow-derived Mesenchymal Cells Research Articles

Precision Delivery of Human Bone Marrow-Derived Mesenchymal Stem Cells Into the Pancreas Via Intra-arterial Injection Prevents the Onset of Diabetes.

Mesenchymal stem cells (MSCs) are a promising therapy to potentially treat diabetes given their potent anti-inflammatory and immune-modulatory properties. While these regenerative cells have shown considerable promise in cell culture, their clinical translation has been challenging. In part, this can be attributed to these cells not reaching the pancreas to exert their regenerative effects following conventional intravenous (IV) injection, with the majority of cells being trapped in the lungs in the pulmonary first-pass effect. In the present study, we will therefore examine whether direct delivery of MSCs to the pancreas via an intra-arterial (IA) injection can improve their therapeutic efficacy. Using a mouse model, in which repetitive low doses of STZ induced a gentle, but progressive, hyperglycemia, we tested bone marrow-derived MSCs (BM-MSCs) which we have shown are enriched with pro-angiogenic and immunomodulatory factors. In cell culture studies, BM-MSCs were shown to preserve islet viability and function following exposure to proinflammatory cytokines (IFN-γ, IL-1β, and TNF-α) through an increase in pAkt. When tested in our animal model, mice receiving IV BM-MSCs were not able to mitigate the effects of STZ, however those which received the same dose and batch of cells via IA injection were able to maintain basal and dynamic glycemic control, to similar levels as seen in healthy control animals, over 10 days. This study shows the importance of considering precision delivery approaches to ensure cell-based therapies reach their intended targets to enable them to exert their therapeutic effects.

Advantages of pooling of human bone marrow-derived mesenchymal stromal cells from different donors versus single-donor MSCs

Mesenchymal stromal cells (MSC) from adult bone marrow are the most commonly used cells in clinical trials. MSCs from single donors are the preferred starting material but suffer from a major setback of being heterogeneous that results in unpredictable and inconsistent clinical outcomes. To overcome this, we developed a method of pooling MSCs from different donors and created cell banks to cater clinical needs. Initially, the master cell banks (MCBs) were created at passage 1 (P1) from the bone marrow MSCs isolated from of nine different donors. At this stage, MCBs from three different donors were mixed in equal proportion and expanded till P3 to create working cell banks. Further, the pooled cells and individual donor MSCs were expanded till P5 and cryopreserved and extensively characterised. There was a large heterogeneity among the individual donor MSCs in terms of growth kinetics (90% Coefficient of variation (CV) for cell yield and 44% CV for population doubling time at P5), immunosuppressive ability (30% CV at 1:1 and 300% CV at 1:10 ratio), and the angiogenic factor secretion potential (20% CV for VEGF and71% CV for SDF-1). Comparatively, the pooled cells have more stable profiles (60% CV for cell yield and 7% CV for population doubling time at P5) and exhibit better immunosuppressive ability (15% CV at 1:1 and 32% CV at 1:10 ratio ) and consistent secretion of angiogenic factors (16% CV for VEGF and 51% CV for SDF-1). Further pooling does not compromise the trilineage differentiation capacity or phenotypic marker expression of the MSCs. The senescence and in vitro tumourigenicity characteristics of the pooled cells are also similar to those of individual donor MSCs. We conclude that pooling of MSCs from three different donors reduces heterogeneity among individual donors and produces MSCs with a consistent secretion and higher immunosuppressive profile.

Bifunctional mesoporous glasses for bone tissue engineering: Biological effects of doping with cerium and polyphenols in 2D and 3D in vitro models

This study evaluates the cytocompatibility of cerium-doped mesoporous bioactive glasses (Ce-MBGs) loaded with polyphenols (Ce-MBGs-Poly) for possible application in bone tissue engineering after tumour resection. We tested MBGs powders and pellets on 2D and 3D in vitro models using human bone marrow-derived mesenchymal stem cells (hMSCs), osteosarcoma cells (U2OS), and endothelial cells (EA.hy926). Promisingly, at a low concentration in culture medium, Poly-loaded MBGs powders containing 1.2 mol% of cerium inhibited U2OS metabolic activity, preserved hMSCs viability, and had no adverse effects on EA.hy926 migration. Moreover, the study discussed the possible interaction between cerium and Poly, influencing anti-cancer effects. In summary, this research provides insights into the complex interactions between Ce-MBGs, Poly, and various cell types in distinct 2D and 3D in vitro models, highlighting the potential of loaded Ce-MBGs for post-resection bone tissue engineering with a balance between pro-regenerative and anti-tumorigenic activities.

Aqueous Supramolecular Transformations of Motor Bola-Amphiphiles at Multiple Length-Scale.

Molecular motor amphiphiles have already been widely attempted for dynamic nanosystems across multiple length-scale for developments of small functional materials, including controlling macroscopic foam properties, amplifying motion as artificial molecular muscles, and serving as extracellular matrix mimicking cell scaffolds. However, limiting examples of bola-type molecular motor amphiphiles are considered for constructing macroscopic biomaterials. Herein, this work presents the designed two second generation molecular motor amphiphiles, motor bola-amphiphiles (MBAs). Aside from the photoinduced motor rotation of MBAs achieved in both organic and aqueous media, the rate of recovering thermal helix inversion step can be controlled by the rotor part with different steric hindrances. Dynamic assembled structures of MBAs are observed under (cryo)-transmission electron microscopy (TEM). This dynamicity assists MBAs in further assembling as macroscopic soft scaffolds by applying a shear-flow method. Upon photoirradiation, the phototropic bending function of MBA scaffolds is observed, demonstrating the amplification of molecular motion into macroscopic phototropic bending functions at the macroscopic length-scale. Since MBAs are confirmed with low cytotoxicity, human bone marrow-derived mesenchymal stem cells (hBM-MSCs) can grow on the surface of MBA scaffolds. These results clearly demonstrate the concept of designing MBAs for developing photoresponsive dynamic functional materials to create new-generation soft robotic systems and cell-material interfaces.

Correlation between Ca Release and Osteoconduction by 3D-Printed Hydroxyapatite-Based Templates.

The application of hydroxyapatite (HA)-based templates is quite often seen in bone tissue engineering since that HA is an osteoconductive bioceramic material, which mimics the inorganic component of mineralized tissues. However, the reported osteoconductivity varies in vitro and in vivo, and the levels of calcium (Ca) release most favorable to osteoconduction have yet to be determined. In this study, HA-based templates were fabricated by melt-extrusion 3D-printing and characterized in order to determine a possible correlation between Ca release and osteoconduction. The HA-based templates were blended with poly(lactide-co-trimethylene carbonate) (PLATMC) at three different HA ratios: 10, 30, and 50%. The printability and physical properties of the HA templates were compared with those of pristine PLATMC. In vitro, osteoconductivity was assessed using seeded human bone marrow-derived mesenchymal stem cells. A mild rate of Ca release was observed for HA10 templates, which exhibited higher mineralized extracellular matrix (ECM) secretion than PLATMC at 14 and 21 days. In contrast, the high rate of Ca release exhibited by HA30 and HA50 templates was associated with reduced osteoconduction and impeded mineralized ECM secretion in vitro. Similar results were observed in vivo. In the calvarial defect model in rabbit, PLATMC and HA10 templates exhibited the highest amount of new bone formation, with obvious contact osteogenesis on their surfaces. In contrast, HA30 and HA50 exhibited distant osteogenesis and reduced amounts of new bone ingrowth. It is concluded that HA-based templates are osteoconductive only at low rates of Ca release.

Plasma-derived extracellular matrix for xenofree and cost-effective organoid modeling for hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) causes significant cancer mortality worldwide. Cancer organoids can serve as useful disease models by high costs, complexity, and contamination risks from animal-derived products and extracellular matrix (ECM) that limit its applications. On the other hand, synthetic ECM alternatives also have limitations in mimicking native biocomplexity. This study explores the development of a physiologically relevant HCC organoid model using plasma-derived extracellular matrix as a scaffold and nutritive biomatrix with different cellularity components to better mimic the heterogenous HCC microenvironment. Plasma-rich platelet is recognized for its elevated levels of growth factors, which can promote cell proliferation. By employing it as a biomatrix for organoid culture there is a potential to enhance the quality and functionality of organoid models for diverse applications in biomedical research and regenerative medicine and to better replicate the heterogeneous microenvironment of HCC.MethodTo generate the liver cancer organoids, HUH-7 hepatoma cells were cultured alone (homogenous model) or with human bone marrow-derived mesenchymal stromal cells and human umbilical vein endothelial cells (heterogeneous model) in plasma-rich platelet extracellular matrix (ECM). The organoids were grown for 14 days and analyzed for cancer properties including cell viability, invasion, stemness, and drug resistance.ResultsHCC organoids were developed comprising HUH-7 hepatoma cells with or without human mesenchymal stromal and endothelial cells in plasma ECM scaffolds. Both homogeneous (HUH-7 only) and heterogeneous (mixed cellularity) organoids displayed viability, cancer hallmarks, and chemoresistance. The heterogeneous organoids showed enhanced invasion potential, cancer stem cell populations, and late-stage HCC genetic signatures versus homogeneous counterparts.ConclusionThe engineered HCC organoids system offers a clinically relevant and cost-effective model to study liver cancer pathogenesis, stromal interactions, and drug resistance. The plasma ECM-based culture technique could enable standardized and reproducible HCC modeling. It could also provide a promising option for organoid culture and scaling up.

A phase I/II clinical trial of ex-vivo expanded human bone marrow derived allogeneic mesenchymal stromal cells in adult patients with perianal fistulizing Crohn’s Disease

BackgroundPerianal fistulas (PF) affect one-third patients with Crohn’s disease (CD) with limited therapeutic options. There is dearth of literature on safety and efficacy of bone marrow-derived mesenchymal stromal cells (BMSCs) in this population.MethodsAn open-label, phase I/II, single-arm study was conducted involving local administration of human allogeneic bone marrow-derived mesenchymal stromal cells in perianal fistula of patients with Crohn’s disease refractory to standard therapies. Clinical severity and biomarkers were assessed at baseline and periodically until week 104 , and MRI at week 24 and 104. Primary and secondary objectives were to assess safety and efficacy respectively. Fistula remission was complete closure of fistula openings with < 2 cm perianal collection on MRI, and fistula response was decrease in drainage by ≥ 50%. Change in perianal disease activity index, quality-of-life and Van Assche index on MRI over time was assessed using mixed-effect linear regression model.ResultsTen patients (male:8, mean age:27.4 ± 12.0years) were recruited. Self-resolving procedure-related adverse events occurred in three patients, with no follow-up adverse events. In intention to treat analysis at week 24, two patients (20%) achieved fistula remission and seven (70%) had fistula response. At week 52, two (20%) patients were in remission and seven (70%) maintained response. At 104 weeks, two (20%) patients maintained response and one (10%) was in remission. Statistically significant decrease in perianal disease activity index (P = 0.008), Van Assche Index (P = 0.008) and improvement in quality-of-life (P = 0.001) were observed over time.ConclusionsAllogeneic BMSCs are safe and effective for the treatment of perianal fistulizing CD with significant improvement in clinical severity and radiological healing.Trial registrationThe study was prospectively registered on Clinical trials registry – India (CTRI), CTRI/2020/01/022743 on 14 January 2020, http://ctri.nic.in.

Gelatin-containing functionally graded calcium sulfate/bioactive glass bone tissue engineering scaffold

One of the challenges and limitations of bone tissue engineering includes fast degradation rates, reduced bioactivity, donor site morbidity, and unresolved risks of pathogen transmission. In the field of bone tissue engineering, gradient materials are promising for treating bone defects because they can create graded structures and compositions similar to natural bone. By controlling the amount of components in the structure, the degradation rate, bioactivity, and osteogenic capacity can be manipulated. In the current study, a gradient, multilayer, porous nanocomposite of calcium sulfate/glass (Gn CS/BG) with a gelatin coating (Gn CS/BG-Gel) was prepared using rotational casting technique. The SEM results showed an average pore size of approximately 400 μm within the Gn CS/BG-Gel nanocomposite. An elastic modulus (E) of around 240 MPa and an ultimate tensile strength (σ) of approximately 5.5 MPa were achieved for Gn CS/BG-Gel nanocomposite. The degradation analysis in a dynamic tris-buffer environment revealed a degradation rate of 68 % over a 63-day period, with a decrease in degradation rate as the bioactive glass (BG) content increased in each layer, indicating the influence of BG on the degradation process. The bioactivity results in a dynamic simulated body fluid suggested that increased BG content in each layer promoted hydroxyapatite formation, indicating improved bioactive behavior. The evaluation of ion release with ICP-OES, MTT and Acridine Orange assays using human bone marrow-derived mesenchymal stem cells (hBMSCs) confirmed the non-toxic nature of Gn CS/BG-Gel. Assessments of alkaline phosphatase (ALP) activity, calcium content, Alizarin Red staining and cell attachment behavior substantiated the osteogenic potential of Gn CS/BG-Gel. Collectively, the three-dimensional structure along with the Gn CS/BG-Gel composition contributed to the enhanced cellular responses. These findings hold significant promise for the development of biomaterials with potential applications in bone tissue engineering, and they contribute valuable insights to the field.

Urine-Derived Stem Cells Reverse Bleomycin‑Induced Experimental Pulmonary Fibrosis by Inhibition of the TGF-β1-Smad2/3 Pathway

BackgroundIdiopathic pulmonary fibrosis (IPF) is characterized by progressive lung interstitial lesions with the disease pathophysiology incompletely understood, which is a serious and fatal disorder with limited treatment options. Mesenchymal stem cells (MSCs) have exhibited promising therapeutic capability for IPF. While most types of MSCs are obtained invasively, urine-derived stem cells (USCs) can be gained in a safe, noninvasive, and inexpensive procedure, which are readily available and reported to exhibit no risk of teratoma formation or oncogenic potential in vivo, sounding alternative to other MSCs. This study aims to investigate the therapeutic effect and mechanism of USCs on IPF, using a bleomycin (BLM)-induced IPF model in mice. MethodsCell surface marker examination by flow cytometry analysis and cell differentiation culture were used to characterize USCs obtained from healthy individuals. BLM was instilled endotracheally in adult C57BL/6 mice, followed by USCs or human bone marrow-derived mesenchymal stem cells (BMSCs) treatment by tail vein injection on day 14. Mice were euthanized on day 14 before administration or day 21 for the evaluation of pulmonary histopathology and hydroxyproline (HYP) content. Inflammatory factors of the lung, including transforming growth factor (TGF)-β1, TNF-α, IL-6, MMP2 were analyzed by quantitative real-time PCR (qRT-PCR). Additionally, immunohistochemistry (IHC) and western blotting (WB) were applied to evaluate the expression of α-SMA and activation of TGF-β1-Smad2/3 in lung. ResultsUSCs highly expressed CD29 and CD90, showing negative expression of hematopoietic stem cell markers (CD45, CD34) and could differentiate into, at least, bone and fat in vitro. In mice challenged with BLM, septal thickening and prominent fibrosis were observed on day 14, with higher HYP content and mRNA levels of TGF-β1, TNF-α and IL-6 exhibited, compared to untreated mice. USCs could migrate to lung and accumulate there in mouse model after intravenous injection. Transplantation of USCs into BLM-induced mice improved their pulmonary histopathology, decreasing Ashcroft score, Szapiel score, HYP content and mRNA levels of TGF-β1 and MMP2 of lung, similar to the effects of BMSCs. IHC and WB further revealed that USCs could inhibit activation of the TGF‑β1-Smad2/3 pathway of lung in vivo. ConclusionsTransplantation of USCs effectively reverses pulmonary fibrotic phenotype in an experimental IPF model, inhibiting the TGF-β1-Smad2/3 pathway, a key driver of fibrosis. These results suggest the therapeutic application of USCs for IPF, instead of other types of MSCs obtained invasively.

Cell Instructive Behavior of Composite Scaffolds in a Co-Culture of Human Mesenchymal Stem Cells and Peripheral Blood Mononuclear Cells.

The in vitro evaluation of 3D scaffolds for bone tissue engineering in mono-cultures is a common practice; however, it does not represent the native complex nature of bone tissue. Co-cultures of osteoblasts and osteoclasts, without the addition of stimulating agents for monitoring cellular cross-talk, remains a challenge. In this study, a growth factor-free co-culture of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and human peripheral blood mononuclear cells (hPBMCs) has been established and used for the evaluation of 3D-printed scaffolds for bone tissue engineering. The scaffolds were produced from PLLA/PCL/PHBV polymeric blends, with two composite materials produced through the addition of 2.5% w/v nanohydroxyapatite (nHA) or strontium-substituted nanohydroxyapatite (Sr-nHA). Cell morphology data showed that hPBMCs remained undifferentiated in co-culture, while no obvious differences were observed in the mono- and co-cultures of hBM-MSCs. A significantly increased alkaline phosphatase (ALP) activity and osteogenic gene expression was observed in co-culture on Sr-nHA-containing scaffolds. Tartrate-resistant acid phosphatase (TRAP) activity and osteoclastogenic gene expression displayed significantly suppressed levels in co-culture on Sr-nHA-containing scaffolds. Interestingly, mono-cultures of hPBMCs on Sr-nHA-containing scaffolds indicated a delay in osteoclasts formation, as evidenced from TRAP activity and gene expression, demonstrating that strontium acts as an osteoclastogenesis inhibitor. This co-culture study presents an effective 3D model to evaluate the regenerative capacity of scaffolds for bone tissue engineering, thus minimizing time-consuming and costly in vivo experiments.

Discovery of YS-1 as a cell line of gastric inflammatory cancer-associated fibroblasts.

Inflammatory cancer-associated fibroblasts (iCAFs) was first identified by co-culture of pancreatic stellate cells and tumor organoids. The key feature of iCAFs is IL-6high/αSMAlow. We examine this phenomenon in gastric cancer using two cell lines of gastric fibroblasts (HGF and YS-1). HGF or YS-1 were co-cultured with MKN7 (a gastric adenocarcinoma cell line) in Matrigel. IL-6 protein levels in the culture supernatant were measured by ELISA. The increased production of IL-6 was not observed in any of the combinations. Instead, the supernatant of YS-1 exhibited the higher levels of IL-6. YS-1 showed IL-6high/αSMA (ACTA2)low in real-time PCR, mRNA-seq and immunohistochemistry. In mRNA-seq, iCAFs-associated genes and signaling pathways were up-regulated in YS-1. No transition to myofibroblastic phenotype was observed by monolayer culture, or the exposure to sonic hedgehog (SHH) or TGF-β. YS-1 conditioned medium induced changes of morphology and stem-ness/differentiation in NUGC-3 (a human gastric adenocarcinoma cell line) and UBE6T-15 (a human bone marrow-derived mesenchymal stem cell line). YS-1 is a stable cell line of gastric iCAFs. This discovery will promote further research on iCAFs for many researchers.

The IL-6 signaling pathway contributes critically to the immunomodulatory mechanism of human decidua-derived mesenchymal stromal cells

Human bone marrow-derived mesenchymal stromal cells (BM-MSCs) have been proposed as a treatment for graft-versus-host disease (GVHD), which is a major complication following allogeneic hematopoietic cell transplantation. However, clinical trials have not yielded good results, and human decidua-derived mesenchymal stromal cells (DSCs) have been proposed as an alternative. In addition, the mechanism by which DSCs exert their immunomodulatory effects is still unknown. We found that knockdown of IL-6 in DSCs reduced the expression of PD-L1 and PD-L2, which are known as classical immune checkpoint inhibitors. Expression of PD-L1 and PD-L2 was restored by adding recombinant IL-6 to the DSCs. When DSCs and IL-6-knockdown DSCs were administered as treatment in a murine GVHD model, the group receiving IL-6-knockdown DSCs had significantly higher mortality and clinical scores compared to the group receiving DSCs. Taken together, these data suggest that the IL-6 signaling pathway is a crucial contributor to the immunosuppressive capacity of DSCs.

A comparative in vitro and in vivo analysis of the impact of copper substitution on the cytocompatibility, osteogenic, and angiogenic properties of a borosilicate bioactive glass.

The 0106-B1-bioactive glass (BG) composition (in wt %: 37.5 SiO2, 22.6 CaO, 5.9 Na2O, 4.0 P2O5, 12.0 K2O, 5.5 MgO, and 12.5 B2O3) has demonstrated favorable processing properties and promising bone regeneration potential. The present study aimed to evaluate the biological effects of the incorporation of highly pro-angiogenic copper (Cu) in 0106-B1-BG in vitro using human bone marrow-derived mesenchymal stromal cells (BMSCs) as well as its in vivo potential for bone regeneration. CuO was added to 0106-B1-BG in exchange for CaO, resulting in Cu-doped BG compositions containing 1.0, 2.5 and 5.0 wt % CuO (composition in wt %: 37.5 SiO2, 21.6/ 20.1/17.6 CaO, 5.9 Na2O, 4.0 P2O5, 12.0 K2O, 5.5 MgO, 12.5 B2O3, and 1.0/ 2.5/ 5.0 CuO). In vitro, the BGs' impact on the viability, proliferation, and growth patterns of BMSCs was evaluated. Analyses of protein secretion, matrix formation, and gene expression were used for the assessment of the BGs' influence on BMSCs regarding osteogenic differentiation and angiogenic stimulation. The presence of Cu improved cytocompatibility, osteogenic differentiation, and angiogenic response when compared with unmodified 0106-B1-BG in vitro. In vivo, a critical-size femoral defect in rats was filled with scaffolds made from BGs. Bone regeneration was evaluated by micro-computed tomography. Histological analysis was performed to assess bone maturation and angiogenesis. In vivo effects regarding defect closure, presence of osteoclastic cells or vascular structures in the defect were not significantly changed by the addition of Cu compared with undoped 0106-B1-BG scaffolds. Hence, while the in vitro properties of the 0106-B1-BG were significantly improved by the incorporation of Cu, further evaluation of the BG composition is necessary to transfer these effects to an in vivo setting.

Modelling Myeloma Dissemination in vitro with hMSC-Interacting Subpopulations of INA-6 Cells and their Aggregation/Detachment Dynamics.

Multiple myeloma involves early dissemination of malignant plasma cells across the bone marrow; however, the initial steps of dissemination remain unclear. Human bone marrow-derived mesenchymal stromal cells (hMSCs) stimulate myeloma cell expansion (e.g., IL-6) and simultaneously retain myeloma cells via chemokines (e.g., CXCL12) and adhesion factors. Hence, we hypothesized that the imbalance between cell division and retention drives dissemination. We present an in vitro model using primary hMSCs co-cultured with INA-6 myeloma cells. Time-lapse microscopy revealed proliferation and attachment/detachment dynamics. Separation techniques (V-well adhesion assay and well plate sandwich centrifugation) were established to isolate MSC-interacting myeloma subpopulations that were characterized by RNAseq, cell viability and apoptosis. Results were correlated with gene expression data (n=837) and survival of myeloma patients (n=536). On dispersed hMSCs, INA-6 saturate hMSC-surface before proliferating into large homotypic aggregates, from which single cells detached completely. On confluent hMSCs, aggregates were replaced by strong heterotypic hMSC-INA-6 interactions, which modulated apoptosis time-dependently. Only INA-6 daughter cells (nMA-INA6) detached from hMSCs by cell division but sustained adherence to hMSC-adhering mother cells (MA-INA6). Isolated nMA-INA6 indicated hMSC-autonomy through superior viability after IL-6 withdrawal and upregulation of proliferation-related genes. MA-INA6 upregulated adhesion and retention factors (CXCL12), that, intriguingly, were highly expressed in myeloma samples from patients with longer overall and progression-free survival, but their expression decreased in relapsed myeloma samples. Altogether, in vitro dissemination of INA-6 is driven by detaching daughter cells after a cycle of hMSC-(re)attachment and proliferation, involving adhesion factors that represent a bone marrow-retentive phenotype with potential clinical relevance.

Microdroplets Encapsulated with NFATc1-siRNA and Exosomes-Derived from MSCs Onto 3D Porous PLA Scaffold for Regulating Osteoclastogenesis and Promoting Osteogenesis.

Osteoporotic-related fractures remains a significant public health concern, thus imposing substantial burdens on our society. Excessive activation of osteoclastic activity is one of the main contributing factors for osteoporosis-related fractures. While polylactic acid (PLA) is frequently employed as a biodegradable scaffold in tissue engineering, it lacks sufficient biological activity. Microdroplets (MDs) have been explored as an ultrasound-responsive drug delivery method, and mesenchymal stem cell (MSC)-derived exosomes have shown therapeutic effects in diverse preclinical investigations. Thus, this study aimed to develop a novel bioactive hybrid PLA scaffold by integrating MDs-NFATc1-silencing siRNA to target osteoclast formation and MSCs-exosomes (MSC-Exo) to influence osteogenic differentiation (MDs-NFATc1/PLA-Exo). Human bone marrow-derived mesenchymal stromal cells (hBMSCs) were used for exosome isolation. Transmission electron microscopy (TEM) and confocal laser scanning microscopy were used for exosome and MDs morphological characterization, respectively. The MDs-NFATc1/PLA-Exo scaffold was fabricated through poly(dopamine) and fibrin gel coating. Biocompatibility was assessed using RAW 264.7 macrophages and hBMSCs. Osteoclast formations were examined via TRAP staining. Osteogenic differentiation of hBMSCs and cytokine expression modulation were also investigated. MSC-Exo exhibited a cup-shaped structure and effective internalization into cells, while MDs displayed a spherical morphology with a well-defined core-shell structure. Following ultrasound stimulation, the internalization study demonstrated efficient delivery of bioactive MDs into recipient cells. Biocompatibility studies indicated no cytotoxicity of MDs-NFATc1/PLA-Exo scaffolds in RAW 264.7 macrophages and hBMSCs. Both MDs-NFATc1/PLA and MDs-NFATc1/PLA-Exo treatments significantly reduced osteoclast differentiation and formation. In addition, our results further indicated MDs-NFATc1/PLA-Exo scaffold significantly enhanced osteogenic differentiation of hBMSCs and modulated cytokine expression. These findings suggest that the bioactive MDs-NFATc1/PLA-Exo scaffold holds promise as an innovative structure for bone tissue regeneration. By specifically targeting osteoclast formation and promoting osteogenic differentiation, this hybrid scaffold may address key challenges in osteoporosis-related fractures.

CpG methylation changes in human mesenchymal and neural stem cells in response to in vitro niche modifications

Stem cell therapies hold promise in addressing the burden of neurodegenerative diseases with human embryonic neural stem cells (hNSC-H9s) and bone marrow-derived human mesenchymal stem cells (hMSCs) as viable candidates. The induction of hMSC neurospheres (hMSC-IN) generate a more lineage-restricted common neural progenitor-like cell population, potentially tunable by heparan sulfate proteoglycans (HSPGs). We examined CpG (5mC) site methylation patterns using Illumina Infinium 850K EPIC arrays in hNSC-H9, hMSCs and hMSC-IN cultures with HSPG agonist heparin at early and late phases of growth. We identified key regulatory CpG sites in syndecans (SDC2; SDC4) that potentially regulate gene expression in monolayers. Unique hMSC-IN hypomethylation in glypicans (GPC3; GPC4) underscore their significance in neural lineages with Sulfatase 1 and 2 (SULF1 &2) CpG methylation changes potentially driving the neurogenic shift. hMSC-INs methylation levels at SULF1 CpG sites and SULF2:cg25401628 were more closely aligned with hNSC-H9 cells than with hMSCs. We further suggest SOX2 regulation governed by lcSOX2-Overall Transcript (lncSOX2-OT) methylation changes with preferential activation of ENO2 over other neuronal markers within hMSC-INs. Our findings illuminate epigenetic dynamics governing neural lineage commitment of hMSC-INs offering insights for targeted mechanisms for regenerative medicine and therapeutic strategies.

Validation of Human Bone Marrow-derived Mesenchymal Stem Cells and MCF-7 Breast Cancer Cells Co-culture Using a 3D Perfused Biomimetic Microfluidic Platform.

Microfluidic experimental models allow to study the mutual interrelation between tumor development and the microvasculature avoiding animal use and lacking interspecies differences. This study aimed to develop and characterize a 3D tissue culture model employing a two-compartment microfluidic chip-perfused platform to visualize and quantify human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and MCF-7 breast cancer cell-cell interactions in real time. MCF-7 cells were implanted in the tumor chamber and hBM-MSCs were injected into microvascular channels. hBM-MSCs culture media was perfused into microvascular compartments. The microfluidic device was microscopically examined weekly for four weeks. VE- and E-cadherin immunofluorescence validated hBM-MSCs differentiation into endothelial cells and MCF-7 cell tumor formation. hBM-MSCs differentiation was highly heterogeneous along the microvascular channels, due to different perfusion flow. hBM-MSCs lining microvascular channels acquired VE-cadherin positive endothelial phenotype and continuously covered microchannels as an endothelium like layer. MCF-7 cells were constantly grown as spheroidal aggregates and later formed a compact area of E-cadherin-positive tumor cells inside tumor compartment. Our study provides valuable knowledge on the properties of hBM-MSCs as vasculogenesis-supporting cells when co-cultured with MCF-7 cells on a 3D perfused biomimetic microfluidic device. This newly established model may serve as an experimental platform for testing anti-tumor/anti-angiogenic drugs.

3D microcapsules for human bone marrow-derived mesenchymal stem cell biomanufacturing in a vertical-wheel bioreactor

3D microcapsules for human bone marrow-derived mesenchymal stem cell biomanufacturing in a vertical-wheel bioreactor

Magnetic colloidal nanoformulations to remotely trigger mechanotransduction for osteogenic differentiation

Nowadays, diseases associated with an ageing population, such as osteoporosis, require the development of new biomedical approaches to bone regeneration. In this regard, mechanotransduction has emerged as a discipline within the field of bone tissue engineering. Herein, we have tested the efficacy of superparamagnetic iron oxide nanoparticles (SPIONs), obtained by the thermal decomposition method, with an average size of 13 nm, when exposed to the application of an external magnetic field for mechanotransduction in human bone marrow-derived mesenchymal stem cells (hBM-MSCs). The SPIONs were functionalized with an Arg-Gly-Asp (RGD) peptide as ligand to target integrin receptors on cell membrane and used in colloidal state. Then, a comprehensive and comparative bioanalytical characterization of non-targeted versus targeted SPIONs was performed in terms of biocompatibility, cell uptake pathways and mechanotransduction effect, demonstrating the osteogenic differentiation of hBM-MSCs. A key conclusion derived from this research is that when the magnetic stimulus is applied in the first 30 min of the in vitro assay, i.e., when the nanoparticles come into contact with the cell membrane surface to initiate endocytic pathways, a successful mechanotransduction effect is observed. Thus, under the application of a magnetic field, there was a significant increase in runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALP) gene expression as well as ALP activity, when cells were exposed to RGD-functionalized SPIONs, demonstrating osteogenic differentiation. These findings open new expectations for the use of remotely activated mechanotransduction using targeted magnetic colloidal nanoformulations for osteogenic differentiation by drug-free cell therapy using minimally invasive techniques in cases of bone loss.

Morinda officinalis polysaccharide promotes the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells via microRNA-210-3p/scavenger receptor class A member 3.

Morinda officinalis polysaccharide (MOP) is the bioactive ingredient extracted from the root of Morinda officinalis, and Morinda officinalis is applied to treat osteoporosis (OP). The purpose of this study was to determine the role of MOP on human bone marrow mesenchymal stem cells (hBMSCs) and the underlying mechanism. HBMSCs were isolated from bone marrow samples of patients with OP and treated with MOP. Quantitative real-time polymerase chain reaction was adopted to quantify the expression of microRNA-210-3p (miR-210-3p) and scavenger receptor class A member 3 (SCARA3) mRNA. Cell Counting Kit-8 assay was employed to detect cell viability; Terminal-deoxynucleotidyl Transferase Mediated Nick End Labeling assay and flow cytometry were adopted to detect apoptosis; Alkaline Phosphatase (ALP) activity assay kit was applied to detect ALP activity; Western blot was executed to quantify the expression levels of SCARA3, osteogenic and adipogenic differentiation markers. Ovariectomized rats were treated with MOP. Bone mineral density (BMD), serum tartrate-resistant acid phosphatase 5b (TRACP 5b), and N-telopeptide of type I collagen (NTx) levels were assessed by BMD detector and Enzyme-linked immunosorbent assay kits. It was revealed that MOP could promote hBMSCs' viability and osteogenic differentiation and inhibit apoptosis and adipogenic differentiation. MOP could also upregulate SCARA3 expression through repressing miR-210-3p expression. Treatment with MOP increased the BMD and decreased the TRACP 5b and NTx levels in ovariectomized rats. MOP may boost the osteogenic differentiation and inhibit adipogenic differentiation of hBMSCs by miR-210-3p/SCARA3 axis.