Expansion Of Mesenchymal Stem Cells Research Articles

Large-Scale Expansion of Umbilical Cord Mesenchymal Stem Cells with Microcarrier Tablets in Bioreactor.

Mesenchymal stem cells show great potential in tissue engineering and cell-based therapies. This protocol demonstrates the use of 3D TableTrix® microcarrier tablets for large-scale manufacturing of human umbilical cord mesenchymal stem cells (hUCMSCs) in a 5-L stirred tank bioreactor. 3D TableTrix® microcarrier tablets readily disperse into tens of thousands of porous microcarriers to simplify cell seeding, and 3D FloTrix® vivaSPIN bioreactor could automate, monitor, and control the entire culture process. 3D TableTrix® microcarriers could also be fully dissolved upon adding dissolution reagent to gently harvest the expanded cells at a high recovery rate. With this protocol, more than 109 cells could be produced in a 5-L bioreactor.

3-D Cell Culture Systems in Bone Marrow Tissue and Organoid Engineering, and BM Phantoms as In Vitro Models of Hematological Cancer Therapeutics—A Review

We review the state-of-the-art in bone and marrow tissue engineering (BMTE) and hematological cancer tissue engineering (HCTE) in light of the recent interest in bone marrow environment and pathophysiology of hematological cancers. This review focuses on engineered BM tissue and organoids as in vitro models of hematological cancer therapeutics, along with identification of BM components and their integration as synthetically engineered BM mimetic scaffolds. In addition, the review details interaction dynamics of various BM and hematologic cancer (HC) cell types in co-culture systems of engineered BM tissues/phantoms as well as their relation to drug resistance and cytotoxicity. Interaction between hematological cancer cells and their niche, and the difference with respect to the healthy niche microenvironment narrated. Future perspectives of BMTE for in vitro disease models, BM regeneration and large scale ex vivo expansion of hematopoietic and mesenchymal stem cells for transplantation and therapy are explained. We conclude by overviewing the clinical application of biomaterials in BM and HC pathophysiology and its challenges and opportunities.

Xenobiotic-Free Medium Guarantees Expansion of Adipose Tissue-Derived Canine Mesenchymal Stem Cells Both in 3D Fibrin-Based Matrices and in 2D Plastic Surface Cultures.

Mesenchymal stem cells (MSCs) have been recently introduced in veterinary medicine as a potential therapeutic tool for several pathologies. The large-scale in vitro expansion needed to ensure the preparation of a suitable number of MSCs for clinical application usually requires the use of xenogeneic supplements like the fetal bovine serum (FBS). The substitution of FBS with species-specific supplements would improve the safety of implanted cells, reducing the risk of undesired immune responses following cell therapy. We have evaluated the effectiveness of canine adipose tissue-derived stromal vascular fraction (SVF) and MSCs (ADMSCs) expansion in the presence of canine blood-derived supplements. Cells were cultured on traditional plastic surface and inside a 3D environment derived from the jellification of different blood-derived products, i.e., platelet-poor plasma (PPP), platelet-rich plasma (PRP), or platelet lysate (PL). PPP, PRP, and PL can contribute to canine ADMSCs in vitro expansion. Both allogeneic and autologous PPP and PL can replace FBS for ADMSCs culture on a plastic surface, exhibiting either a similar (PPP) or a more effective (PL) stimulus to cell replication. Furthermore, the 3D environment based on homospecific blood-derived products polymerization provides a strong stimulus to ADMSCs replication, producing a higher number of cells in comparison to the plastic surface environment. Allogeneic or autologous blood products behave similarly. The work suggests that canine ADMSCs can be expanded in the absence of xenogeneic supplements, thus increasing the safety of cellular preparations. Furthermore, the 3D fibrin-based matrices could represent a simple, readily available environments for effective in vitro expansion of ADMSCs using allogeneic or autologous blood-products.

Influences of Xeno-Free Media on Mesenchymal Stem Cell Expansion for Clinical Application.

Mesenchymal stem/stromal cells (MSCs) are multipotent somatic stem/progenitor cells that can be isolated from various tissues and have attracted increasing attention from the scientific community. This is due to MSCs showing great potential for incurable disease treatment, and most applications of MSCs involve tissue degeneration and treatment of immune- and inflammation-mediated diseases. Conventional MSC cultures contain fetal bovine serum (FBS), which is a common supplement for cell development but is also a risk factor for exposure to animal-derived pathogens. To avoid the risks resulting from the xenogeneic origin and animal-derived pathogens of FBS, xeno-free media have been developed and commercialized to satisfy MSC expansion demands for human clinical applications. This review summarized and provided an overview of xeno-free media that are currently used for MSC expansion. Additionally, we discussed the influences of different xeno-free media on MSC biology with particular regard to cell morphology, surface marker expression, proliferation, differentiation and immunomodulation. The xeno-free media can be serum-free and xeno-free media or media supplemented with some human-originating substances, such as human serum, human platelet lysates, human umbilical cord serum/plasma, or human plasma-derived supplements for cell culture medium. These media have capacity to maintain a spindle-shaped morphology, the expression of typical surface markers, and the capacity of multipotent differentiation and immunomodulation of MSCs. Xeno-free media showed potential for safe use for human clinical treatment. However, the influences of these xeno-free media on MSCs are various and any xeno-free medium should be examined prior to being used for MSC cultures.

Long-term expansion and enhanced osteogenic potential of Macaca MSCs via BMP signaling modulation

Mesenchymal stem cells (MSCs) are a potential source of osteoblasts for the treatment of osteoporosis, but how to better preserve the stemness of MSCs in vitro culture conditions is the main challenge for MSC transplantation. The use of fibroblast growth factor 2 (FGF2) supplement has been described and used extensively to increase the expansion of MSCs. Cumulative evidence indicates that bone morphogenetic protein 2 (BMP2; a member of the TGF-β superfamily) is a secreted protein that promotes bone formation, which can regulate cell growth, differentiation, and development. Here we found that BMP2, in combination with FGF2, not only enhanced the proliferation of Macaca bone marrow-derived MSCs but also strengthened their osteogenic potential after short-term expansion in vitro. During long-term expansion, these cells still retained their osteogenic potential as well as other functional characteristics of pluripotent MSCs, which are gradually lost in the absence of BMP2. In addition, the BMP antagonist Noggin did not affect MSC expansion and the osteogenic potential. This study demonstrates that the regulation of BMP signaling can maintain the effectiveness of MSCs during expansion, which promotes the clinical application of MSCs in bone repair.

Role of Marijuana Component (Cannabidiol) in Induction of Regenerative Ability of Stem Cells

BACKGROUND/PURPOSE: Stem cell therapy has been shown to promote tissue regeneration and wound healing.1,2 However, the isolation and expansion of mesenchymal stem cells can be an invasive, costly, and time-consuming process. Therefore, we sought to improve this process by enhancing the regenerative abilities of stem cells, thereby reducing the quantity of stem cells that must be harvested. Marijuana is a commonly used substance, both recreationally and more recently therapeutically, given its low toxicity and relatively benign side effect profile.3 Certain marijuana components, namely the non-psychoactive cannabidiol (CBD), have been found to function as immunomodulators.4 Our aim was to determine whether CBD-exposed stem cells would demonstrate improved regenerative abilities. MATERIALS/METHODS: Human adipose-derived stem cells (ASC) and bone marrow-derived mesenchymal stem cells (BM) were treated for 6 hours with either low CBD (300 nM) or high CBD (3 mM). A Transwell Migration assay was performed, and absorbance measured at 36 hours. Next, the treated ASC and BM underwent an MTT proliferation assay, with absorbance measured at 36 hours. Finally, a wound healing scratch assay in human keratinocytes was performed, again with ASC treated with low and high dose CBD. RESULTS: Both ASC and BM demonstrated a significant increase in migration with exposure to CBD. Compared to the control, ASC migration increased 412% with low-dose CBD, and 251% with high-dose CBD. For BM, migration increased 298% and 166% with low- and high-dose CBD, respectively. BM demonstrated improved proliferation with exposure to CBD. Compared to the control, proliferation increased 48% with low CBD, and 86% with high CBD (P < 0.05). There were no differences in proliferation in ASCs primed with low or high CBD (P = 0.68). Compared to unexposed ASCs, the low CBD ASC group had 49% faster wound closure at 20 hours and 78% at 44 hours. CONCLUSION: CBD priming of ASCs and BM, at both low and high doses, enhances a number of regeneration parameters, suggesting that this component of marijuana induces or improves stem cell-based therapy. Given the large number of people using various marijuana products, which are largely unregulated and under-studied, these findings may greatly effect management of complex wounds after reconstructive surgery. REFERENCES: 1. Chouhan, D, Dey N, Bhardwaj N, et al. Emerging and innovative approaches for wound healing and skin regeneration: current status and advances. Biomaterials. 2019;216:119267. 2. Hassanshahi A, Hassanshahi M, Khabbazi S, et al. Adipose-derived stem cells for wound healing. J Cell Physiol. 2019;234:7903–7914. 3. Whiting PF, Wolff RF, Deshpande S, et al. Cannabinoids for medical use: a systematic review and meta-analysis. JAMA. 2015;313:2456–2473. 4. Burstein S. Cannabidiol (CBD) and its analogs: a review of their effects on inflammation. Bioorg Med Chem. 2015;23:1377–1385.

Comparison of senescence progression in mesenchymal cells from human umbilical cord walls measured by immunofluorescence and flow cytometry of p16 and p21.

ABSTRACTObjective To follow the expansion of mesenchymal stem cells from umbilical cords by two classic senescence markers, p16 (INK4A) and p21 (CDKN1A), using practical, fast, and less expensive methods than the gold standard Western blotting technique, to evaluate its applicability in the laboratory.Methods Mesenchymal stem cells from umbilical cords were isolated from Wharton’s jelly and, after quality control, morphological and immunophenotypic characterization by flow cytometry, were expanded in culture until coming close to cell cycle arrest (replicative senescence).Results A comparison was made between young cells, at passage 5, and pre-senescent cells, at passage 10, evaluating the protein expression of the classic cell senescence markers p16 and p21, comparing the results obtained by Western blotting with those obtained by flow cytometry and indirect immunofluorescence.Conclusion Follow-up of cell cultures, through indirect p16 immunofluorescence, allows the identification of mesenchymal stem cells from umbilical cord cultures at risk of reaching replicative senescence.

Differentiation Potential of Early- and Late-Passage Adipose-Derived Mesenchymal Stem Cells Cultured under Hypoxia and Normoxia.

With an increasing focus on the large-scale expansion of mesenchymal stem cells (MSCs) required for clinical applications for the treatment of joint and bone diseases such as osteoarthritis, the optimisation of conditions for in vitro MSC expansion requires careful consideration to maintain native MSC characteristics. Physiological parameters such as oxygen concentration, media constituents, and passage numbers influence the properties of MSCs and may have major impact on their therapeutic potential. Cells grown under hypoxic conditions have been widely documented in clinical use. Culturing MSCs on large scale requires bioreactor culture; however, it is challenging to maintain low oxygen and other physiological parameters over several passages in large bioreactor vessels. The necessity to scale up the production of cells in vitro under normoxia may affect important attributes of MSCs. For these reasons, our study investigated the effects of normoxic and hypoxic culture condition on early- and late-passage adipose-derived MSCs. We examined effect of each condition on the expression of key stem cell marker genes POU5F1, NANOG, and KLF4, as well as differentiation genes RUNX2, COL1A1, SOX9, COL2A1, and PPARG. We found that expression levels of stem cell marker genes and osteogenic and chondrogenic genes were higher in normoxia compared to hypoxia. Furthermore, expression of these genes reduced with passage number, with the exception of PPARG, an adipose differentiation marker, possibly due to the adipose origin of the MSCs. We confirmed by flow cytometry the presence of cell surface markers CD105, CD73, and CD90 and lack of expression of CD45, CD34, CD14, and CD19 across all conditions. Furthermore, in vitro differentiation confirmed that both early- and late-passage adipose-derived MSCs grown in hypoxia or normoxia could differentiate into chondrogenic and osteogenic cell types. Our results demonstrate that the minimal standard criteria to define MSCs as suitable for laboratory-based and preclinical studies can be maintained in early- or late-passage MSCs cultured in hypoxia or normoxia. Therefore, any of these culture conditions could be used when scaling up MSCs in bioreactors for allogeneic clinical applications or tissue engineering for the treatment of joint and bone diseases such as osteoarthritis.

SIRT5 deficiency enhances the proliferative and therapeutic capacities of adipose-derived mesenchymal stem cells via metabolic switching.

BackgroundMesenchymal stem cells (MSCs) have therapeutic potential for multiple ischemic diseases. However, in vitro expansion of MSCs before clinical application leads to metabolic reprogramming from glycolysis to oxidative phosphorylation, drastically impairing their proliferative and therapeutic capacities. This study aimed to define the regulatory effects of Sirtuin 5 (SIRT5) on the proliferative and therapeutic functions of adipose‐derived MSCs (ADMSCs) during in vitro expansion.MethodsADMSCs were isolated from wild‐type (WT) and Sirt5‐knockout (Sirt5−/−) mice. Cell counting assay was used to investigate the proliferative capacities of the ADMSCs. Dihydroethidium and senescence‐associated β‐galactosidase stainings were used to measure intracellular ROS and senescence levels. Mass spectrometry was used to analyze protein succinylation. Oxygen consumption rates and extra cellular acidification rates were measured as indicators of mitochondrial respiration and glycolysis. Metabolic‐related genes expression were verified by quantitative PCR and western blot. Hind limb ischemia mouse model was used to evaluate the therapeutic potentials of WT and Sirt5−/− ADSMCs.ResultsSIRT5 protein levels were upregulated in ADMCs during in vitro expansion. Sirt5−/− ADMSCs exhibited a higher proliferation rate, delayed senescence, and reduced ROS accumulation. Furthermore, elevated protein succinylation levels were observed in Sirt5−/− ADMSCs, leading to the reduced activity of tricarboxylic acid cycle‐related enzymes and attenuated mitochondrial respiration. Glucose uptake, glycolysis, and pentose phosphate pathway were elevated in Sirt5−/− ADMSCs. Inhibition of succinylation by glycine or re‐expression of Sirt5 reversed the metabolic alterations in Sirt5−/‐ ADMSCs, thus abolishing their enhanced proliferative capacities. In the hind limb ischemia mouse model, SIRT5−/− ADMSCs transplantation enhanced blood flow recovery and angiogenesis compared with WT ADMSCs.ConclusionsOur results indicate that SIRT5 deficiency during ADMSC culture expansion leads to reversed metabolic pattern, enhanced proliferative capacities, and improved therapeutic outcomes. These data suggest SIRT5 as a potential target to enhance the functional properties of MSCs for clinical application.

Mesenchymal Stem Cell Therapy in Chondral Defects of Knee: Current Concept Review.

Full-thickness cartilage defects if left alone would increase the risk of osteoarthritis (OA) with severe associated pain and functional disability. Articular cartilage defect may result from direct trauma or chronic degeneration. The capability of the mesenchymal stem cells (MSCs) to repair and regenerate cartilage has been widely investigated. This review describes current trends in MSC biology, the sourcing, expansion, application and role of MSCs in chondral defects of human knees. The studies referencing MSCs and knee osteoarthritis were searched (from1998 to 2020) using PubMed, EMBASE, Cochrane Library, Web of Science and theClinicalTrials.gov with keywords (MSCs, chondral defects or cartilage degeneration of knee, cartilage regeneration, chondrogenesis, tissue engineering, efficacy and safety). The inclusion criteria were based on use of MSCs for treatment of chondral defects and osteoarthritis of the knee, English language and human studies. The history of MSC research from the initial discovery of their multipotency to the more recent recognition of theirrole in cartilage defects of knee is elucidated. Several studies have demonstrated promising results in the clinical application for repair of chondral defects as an adjuvant or independent procedure. Intra-articular MSCs provide improvements in pain and function in knee osteoarthritis at short-term follow-up in many studies. The tendency of MSCs to differentiate into fibrocartilage affecting the outcome is a common issue faced by researchers. Some efficacy has been shown of MSCs for cartilage repair in osteoarthritis; however, the evidence of efficacy of intra-articular MSCs on both clinical outcomes and cartilage repair remains limited. Despite the high quality of evidence to support, MSC therapy has emerged but further refinement of methodology will be necessary to support its routine clinical use.

Large-Scale Expansion of Human Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are multipotent stem cells with strong immunosuppressive property that renders them an attractive source of cells for cell therapy. MSCs have been studied in multiple clinical trials to treat liver diseases, peripheral nerve damage, graft-versus-host disease, autoimmune diseases, diabetes mellitus, and cardiovascular damage. Millions to hundred millions of MSCs are required per patient depending on the disease, route of administration, frequency of administration, and patient body weight. Multiple large-scale cell expansion strategies have been described in the literature to fetch the cell quantity required for the therapy. In this review, bioprocessing strategies for large-scale expansion of MSCs were systematically reviewed and discussed. The literature search in Medline and Scopus databases identified 26 articles that met the inclusion criteria and were included in this review. These articles described the large-scale expansion of 7 different sources of MSCs using 4 different bioprocessing strategies, i.e., bioreactor, spinner flask, roller bottle, and multilayered flask. The bioreactor, spinner flask, and multilayered flask were more commonly used to upscale the MSCs compared to the roller bottle. Generally, a higher expansion ratio was achieved with the bioreactor and multilayered flask. Importantly, regardless of the bioprocessing strategies, the expanded MSCs were able to maintain its phenotype and potency. In summary, the bioreactor, spinner flask, roller bottle, and multilayered flask can be used for large-scale expansion of MSCs without compromising the cell quality.

Mesenchymal Stem Cell-Derived Exosomes for Effective Cartilage Tissue Repair and Treatment of Osteoarthritis.

Osteoarthritis (OA) is one of the most common musculoskeletal diseases, caused by cellular inflammatory responses and subsequent structural disruption in osteochondral extracellular matrix (ECM) in cartilage tissues. Various cell-therapies have been recently developed via the exogenous administration of mesenchymal stem cells (MSCs) due to their intrinsic regenerative capacity of self-renewal and chondrogenic differentiation. However, MSC-based cellular approaches have exhibited some technical limitations including dedifferentiation during MSC expansion, reduction of regenerative efficacy upon administration, and inconsistent quality control in large-scale cell production. To overcome these disadvantages, exosome mediated cartilage tissue regeneration has been investigated. Since exosome derived from MSC (MSC-exosome) transport and deliver multiple cellular components from their original MSC sources, they could be utilized as alternative therapeutic agents for OA treatment. Recent studies have shown that the administration of MSC-exosome effectively reduced a production of inflammatory cytokines in chondrocytes, increased the expression of cartilage ECM component, and eventually augment cartilage tissue regeneration in a series of in vivo studies. Therefore, this review emphasizes current engineering approaches via MSC-exosome for cartilage tissue repair, as a functional OA treatment technique.

Aggregation-induced integrated stress response rejuvenates culture-expanded human mesenchymal stem cells.

Protein homeostasis is critical for cellular function, as loss of homeostasis is attributed to aging and the accumulation of unwanted proteins. Human mesenchymal stem cells (MSCs) have shown promising therapeutic potential due to their impressive abilities to secrete inflammatory modulators, angiogenic, and regenerative cytokines. However, there exists the problem of human MSC expansion with compromised therapeutic quality. Duringin vitro expansion, human MSCs are plated on stiff plastics and undergo culture adaptation, which results in aberrant proliferation, shifts in metabolism, and decreased autophagic activity. It has previously been shown that three-dimensional (3D) aggregation can reverse some of these alterations by heightening autophagy and recovering the metabolic state back to a naïve phenotype. To further understand the proteostasis in human MSC culture, this study investigated the effects of 3D aggregation on the human MSC proteome to determine the specific pathways altered by aggregation. The 3D aggregates and 2D cultures of human MSCs derived from bone marrow (bMSC) and adipose tissue (ASC) were analyzed along with differentiated human dermal fibroblasts (FB). The proteomics analysis showed the elevated eukaryotic initiation factor 2 pathway and the upregulated activity of the integrated stress response (ISR) in 3D aggregates. Specific protein quantification further determined that bMSC and ASC responded to ISR, while FB did not. 3D aggregation significantly increased the ischemic survival of bMSCs and ASCs. Perturbation of ISR with small molecules salubrinal and GSK2606414 resulted in differential responses of bMSC, ASC, and FB. This study indicates that aggregation-based preconditioning culture holds the potential for improving the therapeutic efficacy of expanded human MSCs via the establishment of ISR and homeostasis.

Secondary metabolites from acridocarpus orientalis inhibits 4T1 cells and promotes mesenchymal stem cells (MSCs) proliferation.

Among medicinal plants, Acridocarpus orientalis (AO) possesses a remarkable anti-cancer potential, possibly because of its anti-oxidant property. In this study, the leaf and stem extracts from AO were assessed to find the bioactive compound with selective anti-cancer properties. The MTT viability and live and dead assays revealed that around 80% and 98% of 4T1 cells survival were declined after 48h incubation with leaf and stem extracts, respectively. The leaf extract increased stem cell proliferation by 20% whereas the stem extract inhibited around 22% of stem cells proliferation after 48h treatment. The live and dead assay of MSCs confirmed that 40% of the MSCs died when treated with AO stem extract. On the other hand, there were no dead cells after two days of treatment with the leaf extract. Followed by the induction of cell cycle arrest in G0/G1-phase, the real-time PCR demonstrated apoptosis properties in 4T1 cells through overexpression of Bax and down-regulation of BCL2 genes. Interestingly, within the pure compounds isolated from AO leaf extract, Morin was responsible for the inhibition of 4T1 cells proliferation as well as MSCs expansion, predicting to play an essential role in the treatment of cancer. The promisingin vitroanti-cancer and stem cell-inductive properties of morin isolated from AO extract may provide a great potential to produce selective herbal derived drugs.

Enhanced Chondrogenic Capacity of Mesenchymal Stem Cells After TNFα Pre-treatment.

Mesenchymal stem cells (MSCs) are promising cells to treat cartilage defects due to their chondrogenic differentiation potential. However, an inflammatory environment during differentiation, such as the presence of the cytokine TNFα, inhibits chondrogenesis and limits the clinical use of MSCs. On the other hand, it has been reported that exposure to TNFα during in vitro expansion can increase proliferation, migration, and the osteogenic capacity of MSCs and therefore can be beneficial for tissue regeneration. This indicates that the role of TNFα on MSCs may be dependent on the differentiation stage. To improve the chondrogenic capacity of MSCs in the presence of an inflamed environment, we aimed to determine the effect of TNFα on the chondrogenic differentiation capacity of MSCs. Here, we report that TNFα exposure during MSC expansion increased the chondrogenic differentiation capacity regardless of the presence of TNFα during chondrogenesis and that this effect of TNFα during expansion was reversed upon TNFα withdrawal. Interestingly, pre-treatment with another pro-inflammatory cytokine, IL-1β, did not increase the chondrogenic capacity of MSCs indicating that the pro-chondrogenic effect is specific for TNFα. Finally, we show that TNFα pre-treatment increased the levels of SOX11 and active β-catenin suggesting that these intracellular effectors may be useful targets to improve MSC-based cartilage repair. Overall, these results suggest that TNFα pre-treatment, by modulating SOX11 levels and WNT/β-catenin signaling, could be used as a strategy to improve MSC-based cartilage repair.

Cord blood serum harvesting by hydroxyethyl starch: a fetal bovine serum alternative in expansion of umbilical cord-derived mesenchymal stem cells.

As a widely used cell culture supplement, fetal bovine serum (FBS) harbor high content of growth, proliferation, and adhesion factors. However, high cost, bio-safety, possible xenogeneic agent transmission, finite accessible, and ethical controversy are major obstacles that discourage the use of this additive. Accordingly, novel alternatives have been proposed with various pros and cons. Still, caution should be taken in choosing suitable substitute given that the alteration in the main aspects of cultured cells can be biased the consequences of clinical applications. Herein, the authors evaluated the impact of cord blood serum harvesting by hydroxyethyl starch (CBS-HES), as an enriched source of growth factors, on the basic mesenchymal stem cells (MSCs) characteristics. In the present experiment, umbilical cord-derived MSCs were isolated and continuously nourished with Dulbecco's Modified Eagle Medium containing either 10, 15, and 20% CBS-HES or FBSs to compare their morphology, immunophenotype, growth and proliferation rate, death rate, cell cycle, and gene expression profiles. Although all enriched media supported the expansion of MSCs with comparable morphology, cell surface markers, death rate, c-MYC and p16 expression, and growth rate, CBS-HES treated cells significantly (P < 0.05) expressed more hTERT gene in a concentration-dependent manner. Yet no significant shift was observed in the cell cycle of cultured cells using the same concentrations of additives, a finding which further confirmed by Ki-67 immunostaining. CBS-HES as an available and affordable additive, seems to be an optimal, relatively safe, and promising FBS alternative for cultivation, propagation, and subsequent clinical applications of MSCs.

Allogeneic Fibrin Clot for Odontogenic/Cementogenic Differentiation of Human Dental Mesenchymal Stem Cells.

Fetal bovine serum is widely used as a growth supplement for cell culture medium; however, animal-borne pathogens increase the risk of transmitting infectious agents. Platelet-rich fibrin is recently considered as a successful alternative but leukocytes present limits to its allogeneic feasibility. The aim of this study was to explore the effects of allogeneic fibrin clot (AFC) without leukocytes on inducing odontogenic/cementogenic differentiation of human dental pulp stem cells (hDPSCs) and human periodontal ligament stem cells (hPDLSCs) in vitro and in vivo. AFC was prepared by high-speed centrifugation and leukocytes were almost removed, and AFC serum was obtained through three freeze-thaw cycles. hDPSCs and hPDLSCs were treated with AFC serum to investigate the odontogenic or cementogenic associated markers by real-time polymerase chain reaction. hDPSCs were treated with AFC serum and placed inside of dentin canal, hPDLSCs were treated with AFC serum to wrap outside of dentin, the mixture was then transplanted into the subcutaneous of nude mice for 12weeks. AFC serum exhibited enough growth factors and cytokines to induce odontogenic/cementogenic differentiation of hDPSCs and hPDLSCs in vitro. Furthermore, AFC seurum could induce hDPSCs to differentiate into odontoblasts-like cells and pulp-like tissues, and hPDLSCs to regenerate cementum-like tissues. AFC could be an alternative safe source with growth factors for the expansion of human dental mesenchymal stem cells (hDMSCs).

Low Intensity Vibrations Augment Mesenchymal Stem Cell Proliferation and Differentiation Capacity during in vitro Expansion

A primary component of exercise, mechanical signals, when applied in the form of low intensity vibration (LIV), increases mesenchymal stem cell (MSC) osteogenesis and proliferation. While it is generally accepted that exercise effectively combats the deleterious effects of aging in the musculoskeletal system, how long-term exercise affects stem cell aging, which is typified by reduced proliferative and differentiative capacity, is not well explored. As a first step in understanding the effect of long-term application of mechanical signals on stem cell function, we investigated the effect of LIV during in vitro expansion of MSCs. Primary MSCs were subjected to either a control or to a twice-daily LIV regimen for up to sixty cell passages (P60) under in vitro cell expansion conditions. LIV effects were assessed at both early passage (EP) and late passage (LP). At the end of the experiment, P60 cultures exposed to LIV maintained a 28% increase of cell doubling and a 39% reduction in senescence-associated β-galactosidase activity (p < 0.01) but no changes in telomere lengths and p16INK4a levels were observed. Prolonged culture-associated decreases in osteogenic and adipogenic capacity were partially protected by LIV in both EP and LP groups (p < 0.05). Mass spectroscopy of late passage MSC indicated a synergistic decrease of actin and microtubule cytoskeleton-associated proteins in both control and LIV groups while LIV induced a recovery of proteins associated with oxidative reductase activity. In summary, our findings show that the application of long-term mechanical challenge (+LIV) during in vitro expansion of MSCs for sixty passages significantly alters MSC proliferation, differentiation and structure. This suggests LIV as a potential tool to investigate the role of physical activity during aging.

Identifying the Therapeutic Significance of Mesenchymal Stem Cells.

The pleiotropic behavior of mesenchymal stem cells (MSCs) has gained global attention due to their immense potential for immunosuppression and their therapeutic role in immune disorders. MSCs migrate towards inflamed microenvironments, produce anti-inflammatory cytokines and conceal themselves from the innate immune system. These signatures are the reason for the uprising in the sciences of cellular therapy in the last decades. Irrespective of their therapeutic role in immune disorders, some factors limit beneficial effects such as inconsistency of cell characteristics, erratic protocols, deviating dosages, and diverse transfusion patterns. Conclusive protocols for cell culture, differentiation, expansion, and cryopreservation of MSCs are of the utmost importance for a better understanding of MSCs in therapeutic applications. In this review, we address the immunomodulatory properties and immunosuppressive actions of MSCs. Also, we sum up the results of the enhancement, utilization, and therapeutic responses of MSCs in treating inflammatory diseases, metabolic disorders and diabetes.

A bone matrix-simulating scaffold to alleviate replicative senescence of mesenchymal stem cells during long-term expansion.

Replicative senescence during in vitro augmentation, which is mostly induced by the loss of physiological microenvironment, hinders the application of mesenchymal stem cells (MSCs) in the clinic. Here, we investigated whether MSCs senescence could be prevented by bio-scaffold mimicking the natural tissue matrix. Human umbilical cord mesenchymal stem cells (hUCMSCs) exhibited a senescent phenotype during a long-term passage in the conventional culture dish. To fabricate the bone matrix, a naturally based matrix composed of nano-hydroxyapatite/chitosan/poly lactide-co-glycolide (nHA/CS/PLGA) was produced. Long-term passage resulted in an obvious increase in the expression of senescence markers and a reduction in the expression of master genes involved in tissue regeneration. Functional assay confirmed that nHA/CS/PLGA scaffold preserved the proliferation and differentiation of hUCMSCs even after being passaged 27 times. Moreover, in vivo ectopic bone formation assay revealed that the bone formation of hUCMSCs cultured on the nano-scaffolds for the long term was as robust as the cells in the early passage. In summary, our results demonstrate that nHA/CS/PLGA scaffold effectively preserves the stemness and youth of hUCMSCs in the long-term passage. Taken advantage of its compatibility and bioactivity, nHA/CS/PLGA scaffold is of great potential in large-scale expansion of MSCs for stem cell therapy and tissue engineering.