Multipotent Differentiation Capabilities Research Articles

P-793 New approaches for treating diminished ovarian reserve: MSC-derived extracellular vesicles and autologous platelet rich plasma

Abstract Study question Does intraovarian injection of MSC-Derived Extracellular Vesicles (EVs) and autologous platelet rich plasma (PRP) improves the outcome of IVF in women with Diminished Ovarian reserve? Summary answer EVs and PRP therapies increse Antral follicle count (AFC), lower FSH level and result in higher amount of mature oocytes and blastocysts What is known already Diminished ovarian reserve (DOR) and premature ovarian insufficiency (POI) are significant impediments to the success of in vitro fertilization (IVF). Current strategies for ovarian stimulation primarily affects only the growth of antral follicles. Even in scenarios where ovarian ovulatory function is compromised, a reservoir of dormant follicles persists, which could potentially be activated by stem cells. Mesenchymal stem cells (MSCs), owing to their multipotent differentiation capabilities and paracrine signaling properties, have emerged as leading candidates for innovative therapeutic approaches. Recent scientific inquiries have highlighted the paracrine impact of EVs secreted by stem cells, underscoring their prospective therapeutic relevance. Study design, size, duration This prospective interventional study was conducted at the V.I. Kulakov Scientific Research Center for Obstetrics, Gynecology, and Perinatology in Russia. It involved 60 infertile female participants aged 20 to 38 years, each with DOR and a history of at least two unsuccessful IVF cycles. These participants were enrolled in the study during their subsequent IVF cycle. Prior to inclusion, all patients provided written informed consent. Participants/materials, setting, methods Patients in group 1 (n = 30) received treatment with exosomes derived from human umbilical cord mesenchymal stem cells (HUC-MSCs-Exos) prior to initiating their in vitro fertilization (IVF) cycle. In contrast, participants in group 2 (n = 30) underwent intraovarian therapy with autologous platelet-rich plasma (PRP) in preparation for their IVF cycle. The comparative analysis encompassed antral follicle count (AFC), anti-Müllerian hormone (AMH) levels, follicle-stimulating hormone (FSH) levels, ovarian stimulation outcomes, and the primary characteristics of oogenesis and embryogenesis. Main results and the role of chance After transplantation of EVs and PRP the ovarian function-related hormone levels and the AFC returned to nearly normal parameteres.Meanwhile, after exosomal treatment the improvement in reproductive outcomes was more significant. FSH concentration was significantly different before and 30 days after the procedure in both groups. In group 1 FSH values were less than the initial data In 95% of cases, in group 2 – in 72% of cases. AMH levels did not change significantly in both groups. The AFC increased in group 1 compared to group 2. All Patients underwent an IVF antagonist cycle after 3 months from the treatment. The number of obtained oocytes in group 1 ranged from 2 to 8, MII from 1 to 6; in group 2: from 0-5, MII – 0-5. The number of blastocysts in group 1 ranged from 1 to 5, in group 2 – from 0 to 3. In group 1, pregnancy occurred in 9 patients (30%): 1 women ( 3,3%) conceived spontaneously in the cycle of procedures, 8 (26,7%) attempted IVF, in group 2 pregnancy occurred in 4 patients (13,3%): 4 attempted IVF. EVs and PRP injections improve outcomes of IVF results in women with low ovarian reserve. Limitations, reasons for caution Limitations may include the fact that the study is single-center and has a rather small amount of patients enrolled in the study. Fundamental approaches to further research in this area are also required to better understand the obtained results. Wider implications of the findings EVs and PRP therapies appear to enhance ovarian reserve metrics and the success rates of IVF programs in patients with DOR. The clinical implementation of these interventions may offer improved reproductive prospects for patients whose sole option was previously oocyte donation. Trial registration number 121040600410-7

Exploring the Interplay between Bone Marrow Stem Cells and Obesity.

Obesity, a complex disorder with rising global prevalence, is a chronic, inflammatory, and multifactorial disease and it is characterized by excessive adipose tissue accumulation and associated comorbidities. Adipose tissue (AT) is an extremely diverse organ. The composition, structure, and functionality of AT are significantly influenced by characteristics specific to everyone, in addition to the variability connected to various tissue types and its location-related heterogeneity. Recent investigation has shed light on the intricate relationship between bone marrow stem cells and obesity, revealing potential mechanisms that contribute to the development and consequences of this condition. Mesenchymal stem cells within the bone marrow, known for their multipotent differentiation capabilities, play a pivotal role in adipogenesis, the process of fat cell formation. In the context of obesity, alterations in the bone marrow microenvironment may influence the differentiation of mesenchymal stem cells towards adipocytes, impacting overall fat storage and metabolic balance. Moreover, bone marrow's role as a crucial component of the immune system adds another layer of complexity to the obesity-bone marrow interplay. This narrative review summarizes the current research findings on the connection between bone marrow stem cells and obesity, highlighting the multifaceted roles of bone marrow in adipogenesis and inflammation.

Enhancing regenerative medicine: the crucial role of stem cell therapy.

Stem cells offer new therapeutic avenues for the repair and replacement of damaged tissues and organs owing to their self-renewal and multipotent differentiation capabilities. In this paper, we conduct a systematic review of the characteristics of various types of stem cells and offer insights into their potential applications in both cellular and cell-free therapies. In addition, we provide a comprehensive summary of the technical routes of stem cell therapy and discuss in detail current challenges, including safety issues and differentiation control. Although some issues remain, stem cell therapy demonstrates excellent potential in the field of regenerative medicine and provides novel tactics and methodologies for managing a wider spectrum of illnesses and traumas.

Mechanisms of Action of Mesenchymal Stem Cells in Metabolic-Associated Fatty Liver Disease.

Metabolic-associated fatty liver disease (MAFLD) is currently the most common chronic liver disease worldwide. However, its pathophysiological mechanism is complicated, and currently, it has no FDA-approved pharmacological therapies. In recent years, mesenchymal stem cell (MSC) therapy has attracted increasing attention in the treatment of hepatic diseases. MSCs are multipotent stromal cells that originated from mesoderm mesenchyme, which have self-renewal and multipotent differentiation capability. Recent experiments and studies have found that MSCs have the latent capacity to be used for MAFLD treatment. MSCs have the potential to differentiate into hepatocytes, which could be induced into hepatocyte-like cells (HLCs) with liver-specific morphology and function under appropriate conditions to promote liver tissue regeneration. They can also reduce liver tissue injury and reverse the development of MAFLD by regulating immune response, antifibrotic activities, and lipid metabolism. Moreover, several advantages are attributed to MSC-derived exosomes (MSC-exosomes), such as targeted delivery, reliable reparability, and poor immunogenicity. After entering the target cells, MSC-exosomes help regulate cell function and signal transduction; thus, it is expected to become an emerging treatment for MAFLD. In this review, we comprehensively discussed the roles of MSCs in MAFLD, main signaling pathways of MSCs that affect MAFLD, and mechanisms of MSC-exosomes on MAFLD.

Mesenchymal stem/stromal cells in the pathogenesis and regenerative therapy of inflammatory bowel diseases.

Inflammatory bowel diseases (IBDs) represent a group of chronic inflammatory disorders of the gastrointestinal (GI) tract including ulcerative colitis (UC), Crohn’s disease (CD), and unclassified IBDs. The pathogenesis of IBDs is related to genetic susceptibility, environmental factors, and dysbiosis that can lead to the dysfunction of immune responses and dysregulated homeostasis of local mucosal tissues characterized by severe inflammatory responses and tissue damage in GI tract. To date, extensive studies have indicated that IBDs cannot be completely cured and easy to relapse, thus prompting researchers to find novel and more effective therapeutics for this disease. Due to their potent multipotent differentiation and immunomodulatory capabilities, mesenchymal stem/stromal cells (MSCs) not only play an important role in regulating immune and tissue homeostasis but also display potent therapeutic effects on various inflammatory diseases, including IBDs, in both preclinical and clinical studies. In this review, we present a comprehensive overview on the pathological mechanisms, the currently available therapeutics, particularly, the potential application of MSCs-based regenerative therapy for IBDs.

Differentiation Capacity of Human Urine-Derived Stem Cells to Retain Telomerase Activity.

Telomerase activity is essential for the self-renewal and potential of embryonic, induced pluripotent, and cancer stem cells, as well as a few somatic stem cells, such as human urine-derived stem cells (USCs). However, it remains unclear how telomerase activity affects the regeneration potential of somatic stem cells. The objective of this study was to determine the regenerative significance of telomerase activity, particularly to retain cell surface marker expression, multipotent differentiation capability, chromosomal stability, and in vivo tumorigenic transformation, in each clonal population of human primary USCs. In total, 117 USC specimens from 10 healthy male adults (25–57 years of age) were obtained. Polymerase chain reaction amplification of a telomeric repeat was used to detect USCs with positive telomerase activity (USCsTA+). A total of 80 USCsTA+ (70.2%) were identified from 117 USC clones, but they were not detected in the paired normal bladder smooth muscle cell and bone marrow stromal cell specimens. In the 20–40 years age group, approximately 75% of USC clones displayed positive telomerase activity, whereas in the 50 years age group, 59.2% of the USC clones expressed positive telomerase activity. USCsTA+ extended to passage 16, underwent 62.0 ± 4.8 population doublings, produced more cells, and were superior for osteogenic, myogenic, and uroepithelial differentiation compared to USCsTA−. Importantly, USCs displayed normal chromosome and no oncological transformation after being implanted in vivo. Overall, as a safe cell source, telomerase-positive USCs have a robust regenerative potential in cell proliferation and multipotent differentiation capacity.

Metabolic Reconfiguration Activates Stemness and Immunomodulation of PDLSCs.

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

Hypoxia‑induced mitophagy regulates proliferation, migration and odontoblastic differentiation of humandental pulp cells through FUN14 domain‑containing 1.

FUN14 domain-containing 1 (FUNDC1) is a receptor that has been previously reported to activate hypoxia-induced mitophagy. However, the potential role of FUNDC1 in the pathophysiology of dental pulp diseases remains unknown. Therefore, present study first collected tissue specimens from patients with pulpitis and from healthy individuals. The results of reverse transcription-quantitative PCR and immunohistochemical staining revealed markedly increased FUNDC1 and hypoxia-inducible factor-1α expression in pulpitis tissue specimens compared with those from healthy individuals. To provide a theoretical basis for the study of the occurrence, development and reparative mechanisms in the dental pulp after tissue injury, the present study then investigated the role of hypoxia-induced mitophagy in the regulation of proliferation, migration and odontoblastic differentiation in human dental pulp cells (HDPCs), in addition, to the possible involvement of FUNDC1. The surface markers and multipotent differentiation capabilities of HDPCs were performed by flow cytometry (surface markers), alizarin red (osteogenic capabilities), alcian blue (chondrogenic capabilities) and oil red O (adipogenic capabilities). Following culture under hypoxia conditions (1% O2) for varying time periods, the proliferation, migration and odontoblastic differentiation of HDPCs were measured using Cell Counting Kit-8, wound healing and Transwell migration assays, alkaline phosphatase staining and activity tests and western blotting (runt-related transcription factor 2, collagen I, osterix and osteopontin), respectively. Immunofluorescence and western blotting were performed to measure the expression levels of hypoxia-inducible factor-1α, pro-fission dynamin-related protein 1, mitochondria-related proteins translocase of inner mitochondrial membrane 23 and translocase of outer mitochondrial membrane 20, in addition to those of autophagy markers (p62, LC3II, Beclin-1 and autophagy-related 5). Transmission electron microscopy was also used to image the autophagosomes and mitochondrial morphology. In addition, to study the functional role of FUNDC1, its expression was silenced by liposome-mediated transfection with small interfering RNA into HDPCs. Compared with those in HDPCs cultured under normoxic conditions (21% O2), the ability of autophagy in HDPCs cultured under hypoxic conditions for 18 h was markedly increased, whilst the proliferation, migration and odontoblastic differentiation were also enhanced. Increased numbers of autophagosomes could also be observed in the hypoxic group. However, FUNDC1 knockdown in HDPCs reversed the aforementioned effects. Overall, data from the present study suggest that hypoxia can promote the proliferation, migration and odontoblastic differentiation of HDPCs, where the underlying mechanism may be associated with the activation of mitophagy downstream of FUNDC1.

Methods for the Assembly and Characterization of Polyelectrolyte Multilayers as Microenvironments to Modulate Human Mesenchymal Stromal Cell Response.

Thin films are of interest in materials design because they allow for the modification of surface properties of materials while the bulk properties of the material are largely unaffected. In this work, we outline methods for the assembly of thin films using a technique known as layer-by-layer (LbL). Furthermore, their interactions with human mesenchymal stromal cells (hMSCs) are discussed. hMSCs are a subject of growing interest because of their potential to treat or cure diseases, given their immunosuppressive properties, multipotent differentiation capabilities, and tissue regeneration capabilities. Numerous improvements and modifications have been suggested for the harvesting, treatment, and culture of hMSCs prior to their administration in human subjects. Here, we discuss methods to assess the interactions of hMSCs with thin LbL-assembled films of heparin and collagen. Three different methods are discussed. The first details the preparation of heparin/collagen multilayers on different surfaces and the seeding of cells on these multilayers. The second method details the characterization of multilayers, including techniques to assess the thickness, roughness, and surface charge of the multilayers, as well as in situ deposition of multilayers. The third method details the analysis of cell interactions with the multilayers, including techniques to assess proliferation, viability, real-time monitoring of hMSC behavior, analysis of hMSC-adhesive proteins on the multilayers, immunomodulatory factor expression of hMSCs, and cytokine expression on heparin/collagen multilayers. We propose that the methods described in this work will assist in the design and characterization of LbL-assembled thin films and the analysis of hMSCs cultured on these thin films.

A modified protocol for the isolation, culture, and cryopreservation of rat embryonic neural stem cells.

Neural stem cells (NSCs) are characterized by their potential for self-renewal and ability to differentiate into neurons, astrocytes, and oligodendrocytes. They are of great value to scientific studies and clinical applications. Culturing NSCs in vitro is important for characterizing their properties under controlled environmental conditions that may be modified and monitored accurately. The present study explored a modified, detailed and efficient protocol for the isolation, culture and cryopreservation of rat embryonic NSCs. In particular, the viability, nestin expression, and self-renewal and multi-differentiation capabilities of NSCs cryopreserved for various periods of time (7 days, or 1, 6 or 12 months) were characterized and compared. Rat embryonic NSCs were successfully obtained and maintained their self-renewal and multipotent differentiation capabilities even following long-term cryopreservation (for up to 12 months).

A human preadipocyte cell strain with multipotent differentiation capability as an in vitro model for adipogenesis.

Murine 3T3 cell lines constitute a standard model system for in vitro study of mammalian adipogenesis although they do not faithfully reflect the biology of the human adipose cells. Several human adipose cell lines and strains have been used to recapitulate human adipogenesis in vitro, but to date there is no generally accepted in vitro model for human adipogenesis. We obtained a clonal strain of human subcutaneous adipose stromal cells, IPI-SA3-C4, and characterized its utility as an in vitro model for human subcutaneous adipogenesis. IPI-SA3-C4 cells showed a high proliferative potential for at least 30 serial passages, reached 70 cumulative population doublings and exhibited a population doubling time of 47h and colony forming efficiency of 12% at the 57th cumulative population doublings. IPI-SA3-C4 cells remained diploid (46XY) even at the 56th cumulative population doublings and expressed the pluripotency markers POU5F1, NANOG, KLF4, and MYC even at 50th cumulative population doublings. Under specific culture conditions, IPI-SA3-C4 cells displayed cellular hallmarks and molecular markers of adipogenic, osteogenic, and chondrogenic lineages and showed adipogenic capacity even at the 66th cumulative population doublings. These characteristics show IPI-SA3-C4 cells as a promising potential model for human subcutaneous adipogenesis in vitro.

Can mesenchymal stem cells and their conditioned medium assist inflammatory chondrocytes recovery?

Osteoarthritis (OA), one of the most common joint disease, affects more than 80% of the population aged 70 or over. Mesenchymal stem cells (MSCs) show multi-potent differentiation and self-renewal capability, and, after exposure to an inflammatory environment, also exhibit immunosuppressive properties. In this study, we have used a model of lipopolysaccharide (LPS)-stimulated chondrocytes to evaluate MSC anti-inflammatory efficacy. The anti-inflammatory mechanism was tested in two cell-contained culture systems: (i) MSC-chondrocyte indirect contact system and (ii) MSC-chondrocyte direct contact system, and one cytokine-only culture system: MSC-conditioned medium (CM) system. Results showed that MSCs reduced chondrocyte inflammation through both paracrine secretion and cell-to-cell contact. The inflammation-associated, and free-radical-related genes were down-regulated significantly in the direct contact system on 24 h, however, the TNF-α. IL-6 were upregulated and aggrecan, COLII were downregulated on 72 h in direct contact system. Moreover, we found CM produced by MSC possess well therapeutic effect on inflammatory chondorcyte, and the 10-fold concentrated MSC-conditioned medium could down-regulated chondorcyte synthesis inflammation-associated, and free-radical-related genes, such as TNF-α, IL-1β, IL-6 and iNOS even treated for 72 h. In conclusion, MSC-CM showed great potential for MSC-based therapy for OA.

Therapeutic potential of human minor salivary gland epithelial progenitor cells in liver regeneration

Liver disease is a serious problem affecting millions of people with continually increasing prevalence. Stem cell therapy has become a promising treatment for liver dysfunction. We previously reported on human minor salivary gland mesenchymal stem cells (hMSGMSCs), which are highly self-renewable with multi-potent differentiation capability. In this study, keratinocyte-like cells with self-regeneration and hepatic differentiation potential were isolated and characterized, and named human minor salivary gland epithelial progenitor cells (hMSG-EpiPCs). hMSG-EpiPCs were easily obtained via minor intraoral incision; they expressed epithelial progenitor/stem cell and other tissue stem cell markers such as CD29, CD49f, cytokeratins, ABCG2, PLET-1, salivary epithelial cell markers CD44 and CD166, and the Wnt target related gene LGR5 and LGR6. The cells were induced into functional hepatocytes in vitro which expressed liver-associated markers ALB, CYP3A4, AAT, and CK18. Upon transplantation in vivo, they ameliorated severe acute liver damage in SCID mice caused by carbon tetrachloride (CCl4) injection. In a two-thirds partial hepatectomy mouse model, the transplanted cells survived at least 4 weeks and exhibited hepatic potential. These findings demonstrate that hMSG-EpiPCs have potential as a cellular therapy basis for hepatic diseases, physiological and toxicology studies and regenerative medicine.

Sustained Release of Bone Morphogenetic Protein 2 via Coacervate improves Muscle Derived Stem Cell Mediated Cartilage Regeneration in MIA-induced Osteoarthritis

Objectives:Individuals who participate in sports have an increased risk of osteoarthritis (OA), characterized by articular cartilage degeneration. Currently, there is no cure for OA with treatment aimed at symptom relief and improved function. Muscle-derived stem cells (MDSCs) have been shown to exhibit long-term proliferation, high self-renewal, and multipotent differentiation capabilities in vitro. Previously, we have demonstrated that murine MDSCs retrovirally transduced to express chondrogenic proteins (BMPs) differentiate into chondrocytes and enhance cartilage repair in vivo. Direct injection of therapeutic proteins can promote cartilage healing; however, they have relatively short half-lives requiring muitiple injections of high dosages. This presents a challenge in terms of maintaining adequate local BMP levels and could negatively affect both injured and normal structures and lead to side effects such as osteophyte formation. Gene therapy is a promising approach that addresses this problem; however, its utilization in clinical applications is much further down the road. In order to circumvent viral transduction of cells for cartilage regeneration, we developed a unique growth factor delivery platform comprised of native heparin and a synthetic polycation, poly(ethylene argininylaspartate diglyceride) (PEAD) incorporated with BMP2 (BMP2 coacervate). In this study, we show that sustained delivery of BMP2 via a BMP2 coacervate can induce the differentiation of MDSCs to a chondrocyte lineage for in vivo cartilage regeneration and healing in a Monoiodoacetate (MIA)-induced osteoarthritis model.Methods:mMDSCs were isolated from muscle biopsies via a modified pre-plated technique. The BMP2 coacervates were prepared as previously described. The release profiles of BMP2 coacervate were tested by ELISA. The chondrogenic effects that delivery of BMP2 had on MDSCs were evaluated by RT-PCR. The efficacy of MDSC with BMP2 coacervate were evaluated in vivo in a MIA-induced OA model in C57B6 mice. Mice received an intraarticular injection of 20μl of MIA to induce osteoarthritic lesions. Two weeks after MIA injection, mice were injected with PBS (control), MDSC + free BMP2, or MDSC + BMP2 coacervate. Histologic evaluations of cartilage regeneration were conducted at week 4. Institutional Animal Care and Use Committee approval was obtained prior to all animal studies.Results:Release profiles of BMP2 showed sustained release for more than 28 days demonstrating sustained release. The chondrogenic differentiation of MDSCs following BMP2 delivery was assayed using cell culture. The mRNA expression of Aggrecan and Col2A were significantly higher in each BMP2 group compared to control or vehicle only (P<0.05). Multi-dosage free BMP2 demonstrated significantly higher Aggrecan expression compared to single dose free BMP2 (p<0.05). Col2A and aggrecan expression in the BMP coacervate group was superior to both single and mult-dose free BMP2 delivery (p<0.05) (Fig 1A). Histologic examination demonstrated superior cartilage repair and integration in the BMP2 coacervate group (Fig. 1B).Conclusion:This study demonstrates that sustained growth factor delivery (BMP2) is a potential therapeutic option for muscle-derived stem cell based cartilage regeneration for the treatment of osteoarthritis. Our results demonstrate an effective method for prolonged exposure to BMP2 via a non-gene therapy approach which is preferred and clinically translatable.

Effects of adipose-derived stem cells on keloid fibroblasts based on paracrine function

Adipose-derived stem cells (ASCs) possess multipotent differentiation capabilities, including the ability to repair and regenerate injured tissues. Recent studies have reported that ASCs have effects on fibroblasts that are related to wound healing. On the other hand, keloid is a result of pathologic wound healing, and keloid fibroblasts are known to have higher growth potential and an abnormal balance between proliferation and apoptosis. This study aimed to explore the paracrine effects of ASCs on normal fibroblasts (NF) and keloid fibroblasts (KF). NF and KF were cultured alone and in combination with ASCs using a trans-well cell-contact-independent cell culture system. Cell morphology was observed, cell proliferation was assessed using a CCK-8 kit, and apoptosis was assessed by live-dead-cell staining on days 1, 3, and 7. Western blot analysis at 24 hours after culture was used to investigate the production of type 1 collagen and alphasmooth muscle actin (α-SMA). Co-culture with ASCs resulted in increased proliferation of both NF and KF. In the co-culture with ASCs, collagen production decreased and expression of α-SMA increased in both the NF and KF. Comparison between the four groups revealed a significant difference in the expression of both type I collagen and α-SMA (p=0.016 and 0.022; Kruskal-Wallis test). Our findings are different from previous studies that used ASCs for normal dermal fibroblast and normal wound healing. However, considering pathologic mechanisms of keloid, ASCs may have the different effect on keloid fibroblasts, which could be another effect of ASCs modulating keloid during healing process.

FGF8 signaling sustains progenitor status and multipotency of cranial neural crest-derived mesenchymal cells in vivo and in vitro.

The cranial neural crest (CNC) cells play a vital role in craniofacial development and regeneration. They are multi-potent progenitors, being able to differentiate into various types of tissues. Both pre-migratory and post-migratory CNC cells are plastic, taking on diverse fates by responding to different inductive signals. However, what sustains the multipotency of CNC cells and derivatives remains largely unknown. In this study, we present evidence that FGF8 signaling is able to sustain progenitor status and multipotency of CNC-derived mesenchymal cells both in vivo and in vitro. We show that augmented FGF8 signaling in pre-migratory CNC cells prevents cell differentiation and organogenesis in the craniofacial region by maintaining their progenitor status. CNC-derived mesenchymal cells with Fgf8 overexpression or control cells in the presence of exogenous FGF8 exhibit prolonged survival, proliferation, and multi-potent differentiation capability in cell cultures. Remarkably, exogenous FGF8 also sustains the capability of CNC-derived mesenchymal cells to participate in organogenesis such as odontogenesis. Furthermore, FGF8-mediated signaling strongly promotes adipogenesis but inhibits osteogenesis of CNC-derived mesenchymal cells in vitro. Our results reveal a specific role for FGF8 in the maintenance of progenitor status and in fate determination of CNC cells, implicating a potential application in expansion and fate manipulation of CNC-derived cells in stem cell-based craniofacial regeneration.

Osteoprogenitor cells from bone marrow and cortical bone: understanding how the environment affects their fate.

Bone is a dynamic organ where skeletal progenitors and hematopoietic cells share and compete for space. Presumptive mesenchymal stem cells (MSC) have been identified and harvested from the bone marrow (BM-MSC) and cortical bone fragments (CBF-MSC). In this study, we demonstrate that despite the cells sharing a common ancestor, the differences in the structural properties of the resident tissues affect cell behavior and prime them to react differently to stimuli. Similarly to the bone marrow, the cortical portion of the bone contains a unique subset of cells that stains positively for the common MSC-associated markers. These cells display different multipotent differentiation capability, clonogenic expansion, and immunosuppressive potential. In particular, when compared with BM-MSC, CBF-MSC are bigger in size, show a lower proliferation rate at early passages, have a greater commitment toward the osteogenic lineage, constitutively produce nitric oxide as a mediator for bone remodeling, and more readily respond to proinflammatory cytokines. Our data suggest that the effect of the tissue's microenvironment makes the CBF-MSC a superior candidate in the development of new strategies for bone repair.

Endothelial‐mesenchymal transition as a novel mechanism for tissue regeneration

Vascular endothelial cells have shown remarkable plasticity by altering their lineage commitment to generate other cell types to mediate several developmental and pathological processes. We recently found that endothelial cells are converted to a mesenchymal stem cell phenotype through endothelial-mesenchymal transition (EndMT). EndMT has traditionally been implicated in generating the valves and septa of the heart during embryonic development, as well as fibroblast formation in cancer and fibrosis. More recently, we have found that EndMT is a critical mechanism for the formation of endochondral bone during heterotopic ossification, a disabling disorder in which endothelial cells differentiate into chondrocytes and osteocytes through EndMT. Replicating the mechanisms of this disorder may be useful for generating new skeletal tissues for the treatment of degenerative diseases such as osteoarthritis and osteoporosis. Here we describe the EndMT program and discuss the current evidence of EndMT-mediated acquisition of stem cell phenotype and multipotent differentiation capabilities. We suggest that EndMT provides a novel mechanism of using blood vessels to regenerate tissues, rather than traditional stem cell therapies.

A porous membrane-mediated isolation of mesenchymal stem cells from human embryonic stem cells.

Pluripotent human embryonic stem cells (hESCs) acquire mesenchymal characteristics during the epithelial-mesenchymal transition (EMT) process. Here, we report a simple and an efficient isolation method for mesenchymal stem cells (MSCs) from hESCs undergoing EMT using a commercialized porous membrane transwell culture insert. Suspension culture of hESC colonies results in the formation of embryoid bodies, which adhered on the upper compartment of 8 μm porous membrane in the presence of EMG2-MV media. The population migrating through the permeable membrane to the lower compartment not only exhibited EMT markers but also expressed high levels of a panel of typical MSC surface antigen markers, and demonstrated multipotent differentiation capability. In addition, they have a prolonged proliferation capacity without characteristics and chromosomal changes. Furthermore, the isolated MSCs significantly enhanced cardiac functions in a rat model of myocardial infarction (MI) as measured by the left ventricle wall thickness (MI control, 32.9%±3.2% vs. hESCs-MSCs, 38.7%±2.4%), scar length (MI control, 46.1%±2.5% vs. hESCs-MSCs, 41.8%±1.3%), fibrosis area (MI control, 34.3%±1.6% vs. hESCs-MSCs, 28.9%±3.5%), and capillary density. Our findings demonstrate an ease with which hESCs-MSCs can be effectively isolated using the porous membrane, which overcomes the lack of availability of MSCs for therapeutic applications in various diseased animal models.

Plasticity of human dental pulp stromal cells with bioengineering platforms: A versatile tool for regenerative medicine

In recent years, human dental pulp stromal cells (DPSCs) have received growing attention due to their characteristics in common with other mesenchymal stem cells, in addition to the ease with which they can be harvested. In this study, we demonstrated that the isolation of DPSCs from third molar teeth of healthy individuals allowed the recovery of dental mesenchymal stem cells that showed self-renewal and multipotent differentiation capability. DPSCs resulted positive for CD73, CD90, CD105, STRO-1, negative for CD34, CD45, CD14 and were able to differentiate into osteogenic and chondrogenic cells. We also assayed the angiogenic potential of DPSCs, their capillary tube-like formation was assessed using an in vitro angiogenesis assay and the uptake of acetylated low-density lipoprotein was measured as a marker of endothelial function. Based on these results, DPSCs were capable of differentiating into cells with phenotypic and functional features of endothelial cells. Furthermore, this study investigated the growth and differentiation of human DPSCs under a variety of bioengineering platforms, such as low frequency ultrasounds, tissue engineering and nanomaterials. DPSCs showed an enhanced chondrogenic differentiation under ultrasound application. Moreover, DPSCs were tested on different scaffolds, poly(vinyl alcohol)/gelatin (PVA/G) sponges and human plasma clots. We showed that both PVA/G and human plasma clot are suitable scaffolds for adhesion, growth and differentiation of DPSCs toward osteoblastic lineages. Finally, we evaluated the interactions of DPSCs with a novel class of nanomaterials, namely boron nitride nanotubes (BNNTs). From our investigation, DPSCs have appeared as a highly versatile cellular tool to be employed in regenerative medicine.