Maintenance Of Mesenchymal Stem Cells Research Articles

Vascular Cell Adhesion Molecule-1 Regulates Bone Marrow Mesenchymal Stem Cell Maintenance and Niche Function

Bone marrow (BM) mesenchymal stem cells (MSCs) are rare non-hematopoietic perivascular stromal cells characterized by their unique ability to self-renew and differentiate into bone, cartilage, and fat, ensuring proper skeletal development and maintenance. Different subsets of BM MSCs associate with arteriolar and sinusoidal vessels to form specialized niches that regulate hematopoietic stem cell (HSC) function by secreting high levels of niche factors such as CXC-chemokine ligand 12 (CXCL12), stem cell factor (SCF), and Vascular Cell Adhesion Molecule-1 (VCAM1). VCAM1 is classically expressed on endothelial and stromal cells where it acts as an adhesion molecule that preferentially binds to α4β1 integrin on HSCs and progenitor cells (HSPCs). Deletion of Vcam1 in endothelial and hematopoietic cells induces HSPC mobilization into the peripheral blood without affecting endothelial cells homeostasis. Recent work from our lab further demonstrated that VCAM1 expression on HSCs and leukemic stem cells confers innate immune tolerance ( Nat Cell Biol 2022). While the contribution of HSC and endothelial-derived VCAM1 to homeostasis has been well studied, the role of MSC-derived VCAM1 on HSC and MSC maintenance and multilineage potency remains unknown. Our supporting data suggest that MSCs are the BM's main source of Vcam1 (p<0.0001) and that VCAM1 is critical for the maintenance, survival, and function of MSCs. Using a mouse model in which Vcam1 can be conditionally deleted in BM MSCs by a tamoxifen-inducible N-Cadherin-Cre ER line (referred to as Vcam1 N-CadCreER), we found that deletion of Vcam1 significantly reduces the number (4-fold, p=0.0030), frequency (4-fold, p=0.0042), and viability (2-fold, p=0.0039) of BM MSCs. Previous findings in breast cancer revealed that VCAM1 promotes PI3K/Akt survival signaling via Akt phosphorylation. Accordingly, Vcam1 deletion in N-Cadherin-Cre ER MSCs, demonstrated a significant reduction in phospho-Akt (Ser473) (p<0.05), supporting the role of VCAM1 in mediating PI3K/Akt survival signaling in MSCs. Intriguingly, the remaining viable VCAM1-null MSCs isolated from Vcam1 N-CadCreER mice exhibited reduced clonogenic potential (determined by CFU-F assays; 3-fold, p=0.0001). Furthermore, micro-CT analysis of 6-month-old Vcam1 N-CadCreER mice presented an altered bone phenotype compared to the control as evidenced by their significantly increased bone thickness and volume (p<0.05), yet significantly decreased tissue mineral density and medullary cavity area (p<0.05). Bulk RNA sequencing (RNAseq) of sorted viable MSCs revealed distinct grouping between Control and Vcam1 N-CadCreER MSCs. Gene ontology analysis demonstrated that deletion of Vcam1 caused an upregulation of pathways associated with inflammation and cytokine release while pathways associated with ossification, extracellular matrix organization, and skeletal development were all downregulated. Surprisingly, both bulk RNAseq and qPCR analysis confirmed a significant reduction in the expression of HSC niche factors ( Vcam1 Cxcl12 and Scf) following Vcam1 deletion, indicative of reduced niche activity. As expected, Vcam1 deletion in N-Cadherin-Cre ER MSCs, caused significant mobilization of HSCs into the blood (p<0.0001) and BM homing defects (p<0.05), however the number and repopulation capacity of BM HSCs remained unaltered, as seen by BM transplantation experiments. Surprisingly, imaging analysis of whole-mount bone marrow sternum of phenotypic HSCs revealed no differences in HSC niche localization between Vcam1-null and Control mice. Accordingly, ELISA analysis of BM extracellular fluid revealed no differences in overall CXCL12 and SCF levels suggesting that there may be some local or systemic niche compensation at play. Single-cell (sc)RNAseq of pooled BM and compact bone sorted non-hematopoietic stromal cells (DAPI -Lineage -CD45 -Ter119 -CD19 -CD71 -)from Vcam1 N-CadCreERand Control mice further confirmed the downregulation of HSC niche factors in MSCs, and overall remodeling of the BM stromal cell composition following Vcam1 deletion. Interestingly, our scRNAseq analysis revealed increased expression of Cxcl12 and Scf in specific vascular and stromal cell populations, supporting the notion of HSC niche compensation. Overall, these findings demonstrate that MSC-derived Vcam1 is critical for BM MSC survival and their HSC niche function.

Runx2 overexpression promotes bone repair of osteonecrosis of the femoral head (ONFH)

BackgroundRunt-related transcription factor-2 (Runx2) has been considered an inducer to improve bone repair ability of mesenchymal stem cells (MSCs).Methods and resultsTwenty-four rabbits were used to establish Osteonecrosis of the femoral head (ONFH) and randomly devided into four groups: Adenovirus Runx2 (Ad-Runx2) group, Runx2-siRNA group, MSCs group and Model group. At 1 week after model establishment, the Ad-Runx2 group was treated with 5 × 107 MSCs transfected through Ad-Runx2, the Runx2-siRNA group was treated with 5 × 107 MSCs transfected through Runx2-siRNA, the MSCs group was injected with 5 × 107 untreated MSCs, and the Model group was treated with saline. The injection was administered at 1 week and 3 weeks after model establishment. The expression of bone morphogenetic protein 2 (BMP-2), Runx2 and Osterix from the femoral head was detected at 3 and 6 weeks after MSCs being injected, and Masson Trichrome Staining, Gross Morphology, X-ray and CT images observation were used to evaluate the repair effect of ONFH. The data revealed that the expression of BMP-2, Runx2 and Osterix in the Runx2-siRNA group was reduced at 3 weeks compared with the MSCs group, and then the expression further reduced at 6 weeks, but was still higher than the Model group besides Osterix; The expression of these three genes in the Ad-Runx2 group was higher than in the MSCs group. Masson Trichrome Staining, Gross Morphology and X-ray and CT images observation revealed that necrotic femoral head of the MSCs group was more regular and smooth than the Runx2-siRNA group, which has a collapsed and irregular femoral head. In the Ad-Runx2 group, necrotic femoral head was basically completely repaired and covered by rich cartilage and bone tissue.ConclusionsOverexpression of Runx2 can improve osteoblastic phenotype maintenance of MSCs and promote necrotic bone repair of ONFH.

Exosomes derived from hypoxia-preconditioned mesenchymal stem cells (hypoMSCs-Exo): advantages in disease treatment.

Mesenchymal stem cells (MSCs)-based therapy has been reported to be a potential approach to treat various diseases and the paracrine role might be the underlying mechanism. Exosomes were considered an important part of this paracrine role. It was reported that maintenance of MSCs in hypoxia conditions for a short time has shown to be beneficial for the therapeutic effect of MSCs and MSCs-derived exosomes. In this review, we summarized the recent developments on exosomes derived from hypoxia-preconditioned mesenchymal stem cells (hypoMSCs-Exo), including the characteristics of hypoMSCs-Exo in morphology and contents, diseases in which hypoMSCs-Exo showed more effective, and the cellular and molecular mechanisms that hypoMSCs-Exo showed more effective in disease treatment. Besides, we also discussed the limitations of current studies and the issues that needed to be improved in the application of hypoMSCs-Exo. This review aimed to promote a comprehensive and systematic understanding of this type of exosome with great therapeutic potential.

Sensory nerve niche regulates mesenchymal stem cell homeostasis via FGF/mTOR/autophagy axis

Mesenchymal stem cells (MSCs) reside in microenvironments, referred to as niches, which provide structural support and molecular signals. Sensory nerves are niche components in the homeostasis of tissues such as skin, bone marrow and hematopoietic system. However, how the sensory nerve affects the behavior of MSCs remains largely unknown. Here we show that the sensory nerve is vital for mesenchymal tissue homeostasis and maintenance of MSCs in the continuously growing adult mouse incisor. Loss of sensory innervation leads to mesenchymal disorder and a decrease in MSCs. Mechanistically, FGF1 from the sensory nerve directly acts on MSCs by binding to FGFR1 and activates the mTOR/autophagy axis to sustain MSCs. Modulation of mTOR/autophagy restores the MSCs and rescues the mesenchymal tissue disorder of Fgfr1 mutant mice. Collectively, our study provides insights into the role of sensory nerves in the regulation of MSC homeostasis and the mechanism governing it.

Osteogenic differentiation and proliferation potentials of human bone marrow and umbilical cord-derived mesenchymal stem cells on the 3D-printed hydroxyapatite scaffolds

Mesenchymal stem cells (MSCs) are a promising candidate for bone repair. However, the maintenance of MSCs injected into the bone injury site remains inefficient. A potential approach is to develop a bone-liked platform that incorporates MSCs into a biocompatible 3D scaffold to facilitate bone grafting into the desired location. Bone tissue engineering is a multistep process that requires optimizing several variables, including the source of cells, osteogenic stimulation factors, and scaffold properties. This study aims to evaluate the proliferation and osteogenic differentiation potentials of MSCs cultured on 2 types of 3D-printed hydroxyapatite, including a 3D-printed HA and biomimetic calcium phosphate-coated 3D-printed HA. MSCs from bone marrow (BM-MSCs) and umbilical cord (UC-MSCs) were cultured on the 3D-printed HA and coated 3D-printed HA. Scanning electron microscopy and immunofluorescence staining were used to examine the characteristics and the attachment of MSCs to the scaffolds. Additionally, the cell proliferation was monitored, and the ability of cells to differentiate into osteoblast was assessed using alkaline phosphatase (ALP) activity and osteogenic gene expression. The BM-MSCs and UC-MSCs attached to a plastic culture plate with a spindle-shaped morphology exhibited an immunophenotype consistent with the characteristics of MSCs. Both MSC types could attach and survive on the 3D-printed HA and coated 3D-printed HA scaffolds. The MSCs cultured on these scaffolds displayed sufficient osteoblastic differentiation capacity, as evidenced by increased ALP activity and the expression of osteogenic genes and proteins compared to the control. Interestingly, MSCs grown on coated 3D-printed HA exhibited a higher ALP activity and osteogenic gene expression than those cultured on the 3D-printed HA. The finding indicated that BM-MSCs and UC-MSCs cultured on the 3D-printed HA and coated 3D-printed HA scaffolds could proliferate and differentiate into osteoblasts. Thus, the HA scaffolds could provide a suitable and favorable environment for the 3D culture of MSCs in bone tissue engineering. Additionally, biomimetic coating with octacalcium phosphate may improve the biocompatibility of the bone regeneration scaffold.

Tropoelastin improves adhesion and migration of intra-articular injected infrapatellar fat pad MSCs and reduces osteoarthritis progression

Intra-articular injection of mesenchymal stem cells (MSCs) is a promising strategy for osteoarthritis (OA) treatment. However, more and more studies reveal that the injected MSCs have poor adhesion, migration, and survival in the joint cavity. A recent study shows that tropoelastin (TE) regulates adhesion, proliferation and phenotypic maintenance of MSCs as a soluble additive, indicating that TE could promote MSCs-homing in regenerative medicine. In this study, we used TE as injection medium, and compared it with classic media in MSCs intra-articular injection such as normal saline (NS), hyaluronic acid (HA), and platelet-rich plasma (PRP). We found that TE could effectively improve adhesion, migration, chondrogenic differentiation of infrapatellar fat pad MSCs (IPFP-MSCs) and enhance matrix synthesis of osteoarthritic chondrocytes (OACs) in indirect-coculture system. Moreover, TE could significantly enhance IPFP-MSCs adhesion via activation of integrin β1, ERK1/2 and vinculin (VCL) in vitro. In addition, intra-articular injection of TE-IPFP MSCs suspension resulted in a short-term increase in survival rate of IPFP-MSCs and better histology scores of rat joint tissues. Inhibition of integrin β1 or ERK1/2 attenuated the protective effect of TE-IPFP MSCs suspension in vivo. In conclusion, TE promotes performance of IPFP-MSCs and protects knee cartilage from damage in OA through enhancement of cell adhesion and activation of integrin β1/ERK/VCL pathway. Our findings may provide new insights in MSCs intra-articular injection for OA treatment.

Hippo Signaling: A Stress Response Pathway in Stem Cells.

The Hippo pathway, with its core components and the downstream transcriptional coactivators, controls the self-renewable capacity and stemness features of stem cells and serves as a stress response pathway by regulating proliferation, differentiation and apoptosis. The Hippo pathway interaction with other signaling pathways plays an important role in response to various stress stimuli arising from energy metabolism, hypoxia, reactive oxygen species, and mechanical forces. Depending on the energy levels, the Hippo pathway is regulated by AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin (mTOR), which in turn determines stem cell proliferation (cell survival and growth) and differentiation. Oxidative stress-driven by ROS production also affects the Hippo pathway with transcriptional changes through MST/YAP/FoxO pathway and leads to the activation of pro-apoptotic genes and eventually cell death. HIF1alpha/YAP signaling is critical for the long-term maintenance of mesenchymal stem cells (MSCs) under hypoxia. In this review, we present an overview of stem cell response to stress, including mechanical, hypoxia, metabolic and oxidative stress through the modulation of the Hippo pathway. The biological effects such as autophagy, apoptosis and senescence were discussed in the context of the Hippo pathway in stem cells.

Reciprocal interaction between mesenchymal stem cells and transit amplifying cells regulates tissue homeostasis.

Interaction between adult stem cells and their progeny is critical for tissue homeostasis and regeneration. In multiple organs, mesenchymal stem cells (MSCs) give rise to transit amplifying cells (TACs), which then differentiate into different cell types. However, whether and how MSCs interact with TACs remains unknown. Using the adult mouse incisor as a model, we present in vivo evidence that TACs and MSCs have distinct genetic programs and engage in reciprocal signaling cross talk to maintain tissue homeostasis. Specifically, an IGF-WNT signaling cascade is involved in the feedforward from MSCs to TACs. TACs are regulated by tissue-autonomous canonical WNT signaling and can feedback to MSCs and regulate MSC maintenance via Wnt5a/Ror2-mediated non-canonical WNT signaling. Collectively, these findings highlight the importance of coordinated bidirectional signaling interaction between MSCs and TACs in instructing mesenchymal tissue homeostasis, and the mechanisms identified here have important implications for MSC-TAC interaction in other organs.

Influence of olive oil and its components on mesenchymal stem cell biology.

Extra virgin olive oil is characterized by its high content of unsaturated fatty acid residues in triglycerides, mainly oleic acid, and the presence of bioactive and antioxidant compounds. Its consumption is associated with lower risk of suffering chronic diseases and unwanted processes linked to aging, due to the antioxidant capacity and capability of its components to modulate cellular signaling pathways. Consumption of olive oil can alter the physiology of mesenchymal stem cells (MSCs). This may explain part of the healthy effects of olive oil consumption, such as prevention of unwanted aging processes. To date, there are no specific studies on the action of olive oil on MSCs, but effects of many components of such food on cell viability and differentiation have been evaluated. The objective of this article is to review existing literature on how different compounds of extra virgin olive oil, including residues of fatty acids, vitamins, squalene, triterpenes, pigments and phenols, affect MSC maintenance and differentiation, in order to provide a better understanding of the healthy effects of this food. Interestingly, most studies have shown a positive effect of these compounds on MSCs. The collective findings support the hypothesis that at least part of the beneficial effects of extra virgin olive oil consumption on health may be mediated by its effects on MSCs.

Influence of Cell Spreading Area on the Osteogenic Commitment and Phenotype Maintenance of Mesenchymal Stem Cells

Osteogenic differentiation and commitment of mesenchymal stem cells (MSCs) is a complex process that is induced and regulated by various biological factors and biophysical cues. Although cell spreading area, as a biophysical cue, has been demonstrated to play a critical role in the regulation of osteogenic differentiation of MSCs, it is unclear how it affects the maintenance of the committed phenotype after osteogenic differentiation of MSCs. In this study, poly (vinyl alcohol) was micropatterned on a tissue culture polystyrene surface, and the micropatterns were used to culture MSCs to control their cell spreading area. The influence of cell spreading area on osteogenic differentiation and maintenance of the differentiated phenotype of MSCs was investigated. MSCs with a larger spreading area showed a higher degree of osteogenic differentiation, slower loss of differentiated phenotype and slower re-expression of stem cell markers compared with MSCs with a smaller spreading area. A large cell spreading area was beneficial for osteogenic differentiation of MSCs and maintenance of their differentiated phenotype.

Defined Substrate by Aptamer Modification with the Balanced Properties of Selective Capture and Stemness Maintenance of Mesenchymal Stem Cells.

The recruitment of endogenous mesenchymal stem cells (MSCs), as an alluring approach for in situ tissue regeneration, always accompanies with other types of cells. Therefore, it is of enormous value to bestow a substrate with the property of selective capture to MSCs. However, it was reported that when MSCs are cultured on a substrate with excessive affinity, their stemness diminished. Therefore, constructing a substrate with the balanced ability of selective capture and stemness maintenance becomes a big challenge. In this study, an Aptamer 19S (Apt19S)-modified substrate was fabricated by grafting Apt19S on a PEGylated glass substrate. The X-ray photoelectron spectroscopy results verified that the antifouling poly(ethylene glycol) (PEG) layer was created. Tracking by ellipsometry, the thicknesses of PEG layers were proved to increase with PEG concentration. The results of the quartz crystal microbalance also validated that the Apt19S densities could be modulated by the concentrations of the Apt19S solution. The results of the cell adhesion assay indicated that the modification of Apt19S caused a significant increase in the adhesion ratio and area of rBMSCs. Selective adhesion was confirmed by coculture of rBMSCs with macrophages and NIH3T3 cells, demonstrating that a higher proportion of rBMSCs adhered to the Apt19S-modified substrate. The results of specific capture were further confirmed by a flow model to simulate the body fluid flow. The comprehensive results of reverse transcription polymerase chain reaction, immunofluorescence staining, proliferation capacity, and differentiation assay showed that the stemness of rBMSCs was maintained better on a substrate with the appropriate Apt19S density. All of these results indicated that Apt19S modification is an effective strategy to endow a substrate with the specific capture ability of MSCs, and the balance between selective capture and stemness maintenance can be achieved by the precise regulation of the aptamer density.

Combined Single-Cell and Spatial Transcriptomics to Deconvolute the Hematopoietic Stem Cell Niche

Coordinated interaction of many cell types is required to facilitate hematopoietic and mesenchymal stem cell maintenance and differentiation in the bone marrow. However, the molecular factors and cell types involved in this complex interplay remain poorly understood. Here we developed a combined single cell and spatial transcriptomics approach to address this problem.Large-scale single-cell transcriptional profiling in conjunction with a multi-layered sorting approach allowed us to generate a complete and evenly sampled transcriptional map of all major bone and bone marrow populations. Our dataset covers all cell types or differentiation trajectories involved in mesenchymal and hematopoietic stem cell differentiation, osteogenesis, adipogenesis, myelopoiesis, erythropoiesis, lymphopoiesis, memory T cell formation as well as bone marrow neural innervation and vascularization at the single cell level. Using this data, we derive fundamental properties of the described cell types, clarify the cellular source of signals affecting stem cell differentiation processes and provide a systems view on putative intercellular interactions. Systematic spatial transcriptomics, using laser-capture microdissection of selected bone marrow niches followed by transcriptional profiling and bioinformatic cellular deconvolution, allowed us to confirm predicted interactions and map the cellular composition of distinct bone marrow niches.Our analyses highlight the importance of pre-adipogenic CXCL12 abundant reticular cells as key niche cells for stem cell maintenance, provides a holistic systems view of the hematopoietic stem cell niche and offers a novel approach to systematically deconvolute the molecular, cellular and spatial composition of complex tissues. DisclosuresNo relevant conflicts of interest to declare.

Long non-coding RNA HULC affects the proliferation, apoptosis, migration, and invasion of mesenchymal stem cells.

Exploring the molecular mechanisms of growth and function in MSCs is the key to improve their clinical therapeutic effects. Currently, more and more evidence show that the long non-coding RNA (lncRNA) plays an important role in the growth, stemness and function of MSCs.Both HULC and MALAT1 are the earliest discovered LNCRNAs, which are closely related to tumor growth. All of them can promote the growth of liver cancer stem cells. Previously, we have studied the effects of MALAT1 on the growth and function of MSCs. In this study, we focused on the effects of HULC on MSCs. We elucidated the effects of HULC on the growth and differentiation of MSCs, and explored the relationship between inflammatory stimuli and HULC expression in MSCs. Our findings provide a new molecular target for the growth and clinical application of MSCs.

RETRACTED ARTICLE: Platelet-derived growth factor receptor-α and -β promote cancer stem cell phenotypes in sarcomas

Sarcomas are malignant tumors derived from mesenchymal tissues and may harbor a subset of cells with cancer stem-like cell (CSC) properties. Platelet-derived growth factor receptors α and β (PDGFR-α/β) play an important role in the maintenance of mesenchymal stem cells. Here we examine the role of PDGFR-α/β in sarcoma CSCs. PDGFR-α/β activity and the effects of PDGFR-α/β inhibition were examined in 3 human sarcoma cell lines using in vitro assays and mouse xenograft models. In all three cell lines, PDGFR-α/β activity was significantly higher in cells grown as spheroids (to enrich for CSCs) and in cells sorted for CD133 expression (a marker of sarcoma CSCs). Self-renewal transcription factors Nanog, Oct4, and Slug and epithelial-to-mesenchymal transition (EMT) proteins Snail, Slug, and Zeb1 were also significantly higher in spheroids cells and CD133(+) cells. Spheroid cells and CD133(+) cells demonstrated 2.9- to 4.2-fold greater migration and invasion and resistance to doxorubicin chemotherapy. Inhibition of PDGFR-α/β in CSCs using shRNA or pharmacologic inhibitors reduced expression of certain self-renewal and EMT proteins, reduced spheroid formation by 74–82%, reduced migration and invasion by 73–80%, and reversed chemotherapy resistance. In mouse xenograft models, combining PDGFR-α/β inhibition (using shRNA or imatinib) with doxorubicin had a more-than-additive effect in blocking tumor growth, with enhanced apoptosis, especially in CD133(+) cells. These results indicate that PDGFR-α/β activity is upregulated in sarcoma CSCs and promote CSC phenotypes including migration, invasion, and chemotherapy resistance. Thus, the PDGFR-α/β pathway represents a new potential therapeutic target to reduce metastatic potential and increase chemosensitivity.

Optimization of Alginate-Based Encapsulation Utilization For Viability and Stability of The Mesenchymal Stem Cell

In the past few decades, attention and research in the field of stem cell are progressing very rapidly. Hospitals in Indonesia have been using stem cells as an alternative to cure some illnesses like diabetes, heart disease, fractures and joints, dental implants, etc. Currently, adult stem cells can be obtained not only from the spinal cord and peripheral vessels, but also from fat tissues of the human body, where it can be isolated as adherent stem cells (mesenchymal stem cells). Consideration of fat tissue as the source of mesenchymal stem cells (MSCs) for autologous tissue engineering is because they are readily available in abundant quantities through minimal invasive procedures, as well as easily cultured and propagated. It is possible to proliferate and differentiate into the desired direction of the network. Stem cell growth requires conditions to grow such as requiring optimum growing conditions such as an environmental temperature of 37°C and a concentration of 5% CO2. Maintenance of MSCs also requires a subculture process, i.e. the process of moving MSCs from a full culture medium to new media; continuous subculture process can cause changes in MSCs. The viability of stem cells may be disrupted by micro-conditions in wounds such as hypoxia, oxidative stress, and inflammation. Therefore, the purpose of this research was to investigate whether alginate-based encapsulation can increase and maintenance stem cell growth at different temperature by using some concentration of alginate and CaCl2 as the formula. Results shown that alginat with low concentration and CaCl2 100mM is suitable for MSCs growth (as in MTT result shown) at 25°C temperature. This can be due to the MSCs encapsulated can adapt and grow within the alginate microcapsule with low concentration. In addition, the media may also easier to get into the microcapsule alginate.

Collagen-Based Medical Device as a Stem Cell Carrier for Regenerative Medicine

Maintenance of mesenchymal stem cells (MSCs) requires a tissue-specific microenvironment (i.e., niche), which is poorly represented by the typical plastic substrate used for two-dimensional growth of MSCs in a tissue culture flask. The objective of this study was to address the potential use of collagen-based medical devices (HEMOCOLLAGENE®, Saint-Maur-des-Fossés, France) as mimetic niche for MSCs with the ability to preserve human MSC stemness in vitro. With a chemical composition similar to type I collagen, HEMOCOLLAGENE® foam presented a porous and interconnected structure (>90%) and a relative low elastic modulus of around 60 kPa. Biological studies revealed an apparently inert microenvironment of HEMOCOLLAGENE® foam, where 80% of cultured human MSCs remained viable, adopted a flattened morphology, and maintained their undifferentiated state with basal secretory activity. Thus, three-dimensional HEMOCOLLAGENE® foams present an in vitro model that mimics the MSC niche with the capacity to support viable and quiescent MSCs within a low stiffness collagen I scaffold simulating Wharton’s jelly. These results suggest that haemostatic foam may be a useful and versatile carrier for MSC transplantation for regenerative medicine applications.

The involvement of histone methylation in osteoblastic differentiation of human periosteum-derived cells cultured in vitro under hypoxic conditions.

Although oxygen concentrations affect the growth and function of mesenchymal stem cells (MSCs), the impact of hypoxia on osteoblastic differentiation is not understood. Likewise, the effect of hypoxia-induced epigenetic changes on osteoblastic differentiation of MSCs is unknown. The aim of this study was to examine the in vitro hypoxic response of human periosteum-derived cells (hPDCs). Hypoxia resulted in greater proliferation of hPDCs as compared with those cultured in normoxia. Further, hypoxic conditions yielded decreased expression of apoptosis- and senescence-associated genes by hPDCs. Osteoblast phenotypes of hPDCS were suppressed by hypoxia, as suggested by alkaline phosphatase activity, alizarin red-S-positive mineralization, and mRNA expression of osteoblast-related genes. Chromatin immunoprecipitation assays showed an increased presence of H3K27me3, trimethylation of lysine 27 on histone H3, on the promoter region of bone morphogenetic protein-2. In addition, mRNA expression of histone lysine demethylase 6B (KDM6B) by hPDCs was significantly decreased in hypoxic conditions. Our results suggest that an increased level of H3K27me3 on the promoter region of bone morphogenetic protein-2, in combination with downregulation of KDM6B activity, is involved in the suppression of osteogenic phenotypes of hPDCs cultured in hypoxic conditions. Although oxygen tension plays an important role in the viability and maintenance of MSCs in an undifferentiated state, the effect of hypoxia on osteoblastic differentiation of MSCs remains controversial. In addition, evidence regarding the importance of epigenetics in regulating MSCs has been limited. This study was to examine the role hypoxia on osteoblastic differentiation of hPDCs, and we examined whether histone methylation is involved in the observed effect of hypoxia on osteogenic differentiation of hPDCs.

Hyaluronan keeps mesenchymal stem cells quiescent and maintains the differentiation potential over time.

SummaryHyaluronan (HA), an abundant polysaccharide found in human bodies, plays a role in the mesenchymal stem cells (MSCs) maintenance. We had previously found that HA prolonged the lifespan, and prevented the cellular aging of murine adipose‐derived stromal cells. Recently, we had also summarized the potential pathways associated with HA regulation in human MSCs. In this study, we used the human placenta‐derived MSCs (PDMSC) to investigate the effectiveness of HA in maintaining the PDMSC. We found that coating the culture surface coated with 30 μg cm−2 of HA (C) led to cluster growth of PDMSC, and maintained a higher number of PDMSC in quiescence compared to those grown on the normal tissue culture surface (T). PDMSC were treated for either 4 (short‐term) or 19 (long‐term) consecutive passages. PDMSC which were treated with HA for 19 consecutive passages had reduced cell enlargement, preserved MSCs biomarker expressions and osteogenic potential when compared to those grown only on T. The PDMSC transferred to T condition after long‐term HA treatment showed preserved replicative capability compared to those on only T. The telomerase activity of the HA‐treated PDMSC was also higher than that of untreated PDMSC. These data suggested a connection between HA and MSC maintenance. We suggest that HA might be regulating the distribution of cytoskeletal proteins on cell spreading in the event of quiescence to preserve MSC stemness. Maintenance of MSCs stemness delayed cellular aging, leading to the anti‐aging phenotype of PDMSC.

Three‐dimensional carbon nanotube scaffolds for long‐term maintenance and expansion of human mesenchymal stem cells

Expansion of mesenchymal stem cells (MSCs) and maintenance of their self-renewal capacity in vitro requires specialized robust cell culture systems. Conventional approaches using animal-derived or artificial matrices and a cocktail of growth factors have limitations such as consistency, scalability, pathogenicity, and loss of MSC phenotype. Herein, we report the use of all-carbon 3-D single- and multiwalled carbon nanotube scaffolds (SWCNTs and MWCNTs) as artificial matrices for long-term maintenance and expansion of human MSCs. Three-dimensional SWCNT and MWCNT scaffolds were fabricated using a novel radical initiated thermal cross-linking method that covalently cross-links CNTs to form 3-D macroporous all-carbon architectures. Adipose-derived human MSCs showed good cell viability, attachment, proliferation, and infiltration in MWCNT and SWCNT scaffolds comparable to poly(lactic-co-glycolic) acid (PLGA) scaffolds (baseline control). ADSCs retained stem cell phenotype after 30 days and satisfied the International Society for Cellular Therapy's (ISCT) minimal criteria for MSCs. Post expansion, (1) ADSCs showed in vitro adherence to tissue culture polystyrene (TCPS); (2) MSC surface antigen expression [CD14(-), CD19(-), CD34(-), CD45(-), CD73(+), CD90(+), CD105(+)]; and (3) trilineage differentiation into osteoblasts, adipocytes, and chondrocytes. Results show that cross-linked 3-D MWCNTs and SWCNTs scaffolds are suitable for ex vivo expansion and maintenance of MSCs for therapeutic applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1927-1939, 2017.

Comparison of MSC-Neurogenin1 administration modality in MCAO rat model.

Intracerebral (IC) grafting of mesenchymal stem cells (MSCs) is not currently used in humans due to its potential complications. On the other hand, intra-arterial (IA) administration can be facilitated for engrafting of intensifed MSCs in the injured human brain. The study is designed to compare the two methods of MSC administration using IA and IC routes through the parameters of behavior, infarct volume, cell distribution, and MSC identification. An ischemic stroke model was generated in Sprague Dawley male rats. This experiment used MSCs/Ngn1 that express Neurogenin1 (Ngn1) to ensure grafted MSC maintenance. MSCs/Ngn1 or normal saline was administrated via the IC or IA route on day 3. All animals were randomly assigned into four groups (five rats in each group): IC-control, IA-control, IC-MSCs/Ngn1, or IA-MSCs/Ngn1. Motor behaviors, infarct volume, and distribution of superparamagnetic iron oxide (SPIO)-labeled cells on magnetic resonance imaging (MRI) were compared from each group. There were no baseline differencess in motor behaviors or infarct volume between IC-MSCs/Ngn1 and IA-MSCs/Ngn1. Hovever, the IA-MSCs/Ngn1 group showed the greatest recovery on Rotarod testing and adhesive removal tests (p = 0.003 and p = 0.009 vs. IC-MSCs/Ngn1, respectively). The IA-MSCs/Ngn1 group also had more evenly distributed SPIO-labeled cells on MRI. The results suggest that IA administration is likely to be benefcial for humans based on its ability to improve behavioral outcomes and ensure even MSC engrafting.