Marrow-derived Mesenchymal Cells Research Articles

Mesenchymal cells support the early retention of primary alveolar type 2 cells on acellular mouse lung scaffolds

ObjectivesTissue engineering approaches via repopulation of acellular biological grafts provide an exciting opportunity to generate lung grafts for transplantation. Alveolar type 2 (AT2) cells are a promising cell source for re-epithelialization. There are however inherent limitations with respect to their survival and growth, thus impeding their usability for tissue engineering applications. This study investigates the use of mesenchymal stromal cells to support primary AT2 cells for recellularization of mouse lung scaffolds. MethodsAT2 cells and bone marrow-derived mesenchymal cells (BMC) were co-delivered to decellularized mouse lung scaffolds. Recellularized lungs were evaluated for cell surface coverage, viability, and differentiation at 1 and 4 days after cell seeding. Recellularization was evaluated via histological analysis and immunofluorescence. ResultsSimultaneous delivery of AT2 and BMC into acellular lung scaffolds resulted in enhanced cell surface coverage and reduced AT2 cell apoptosis in the recellularized scaffolds at Day 1 but not Day 4. AT2 cell number decreased after 4 days in both of AT2 only and codelivery groups suggesting limited expansion potential in the scaffold. After retention in the scaffold, AT2 cells differentiated into Aqp5-expressing cells. ConclusionsOur results indicate that BMC support AT2 cell survival during the initial attachment and engraftment phase of recellularization. While our findings suggest only a short-term beneficial effect of BMC, our study demonstrates that AT2 cells can be delivered and retained in acellular lung scaffolds; thus with preconditioning and supporting cells, may be used for re-epithelialization. Selection and characterization of appropriate cell sources for use in recellularization, will be critical for ultimate clinical application.

Multipotent Mesenchymal Cells Homing and Differentiation on Poly(ε-caprolactone) Blended with 20% Tricalcium Phosphate and Polylactic Acid Incorporating 10% Hydroxyapatite 3D-Printed Scaffolds via a Commercial Fused Deposition Modeling 3D Device.

As highlighted by the 'Global Burden of Disease Study 2019' conducted by the World Health Organization, ensuring fair access to medical care through affordable and targeted treatments remains crucial for an ethical global healthcare system. Given the escalating demand for advanced and urgently needed solutions in regenerative bone procedures, the critical role of biopolymers emerges as a paramount necessity, offering a groundbreaking avenue to address pressing medical needs and revolutionize the landscape of bone regeneration therapies. Polymers emerge as excellent solutions due to their versatility, making them reliable materials for 3D printing. The development and widespread adoption of this technology would impact production costs and enhance access to related healthcare services. For instance, in dentistry, the use of commercial polymers blended with β-tricalcium phosphate (TCP) is driven by the need to print a standardized product with osteoconductive features. However, modernization is required to bridge the gap between biomaterial innovation and the ability to print them through commercial printing devices. Here we showed, for the first time, the metabolic behavior and the lineage commitment of bone marrow-derived multipotent mesenchymal cells (MSCs) on the 3D-printed substrates poly(e-caprolactone) combined with 20% tricalcium phosphate (PCL + 20% β-TCP) and L-polylactic acid (PLLA) combined with 10% hydroxyapatite (PLLA + 10% HA). Although there are limitations in printing additive-enriched polymers with a predictable and short half-life, the tested 3D-printed biomaterials were highly efficient in supporting osteoinductivity. Indeed, considering different temporal sequences, both 3D-printed biomaterials resulted as optimal scaffolds for MSCs' commitment toward mature bone cells. Of interest, PLLA + 10% HA substrates hold the confirmation as the finest material for osteoinduction of MSCs.

Extracellular Vesicles Derived from Osteogenic-Differentiated Human Bone Marrow-Derived Mesenchymal Cells Rescue Osteogenic Ability of Bone Marrow-Derived Mesenchymal Cells Impaired by Hypoxia.

In orthopedics, musculoskeletal disorders, i.e., non-union of bone fractures or osteoporosis, can have common histories and symptoms related to pathological hypoxic conditions induced by aging, trauma or metabolic disorders. Here, we observed that hypoxic conditions (2% O2) suppressed the osteogenic differentiation of human bone marrow-derived mesenchymal cells (hBMSC) in vitro and simultaneously increased reactive oxygen species (ROS) production. We assumed that cellular origin and cargo of extracellular vesicles (EVs) affect the osteogenic differentiation capacity of hBMSCs cultured under different oxygen pressures. Proteomic analysis revealed that EVs isolated from osteogenic differentiated hBMSC cultured under hypoxia (hypo-osteo EVs) or under normoxia (norm-osteo EVs) contained distinct protein profiles. Extracellular matrix (ECM) components, antioxidants and pro-osteogenic proteins were decreased in hypo-osteo EVs. The proteomic analysis in our previous study revealed that under normoxic culture conditions, pro-osteogenic proteins and ECM components have higher concentrations in norm-osteo EVs than in EVs derived from naïve hBMSCs (norm-naïve EVs). When selected for further analysis, five anti-hypoxic proteins were significantly upregulated (response to hypoxia) in norm-osteo EVs. Three of them are characterized as antioxidant proteins. We performed qRT-PCR to verify the corresponding gene expression levels in the norm-osteo EVs' and norm-naïve EVs' parent cells cultured under normoxia. Moreover, we observed that norm-osteo EVs rescued the osteogenic ability of naïve hBMSCs cultured under hypoxia and reduced hypoxia-induced elevation of ROS production in osteogenic differentiated hBMSCs, presumably by inducing expression of anti-hypoxic/ antioxidant and pro-osteogenic genes.

Inhibition of Gap Junction Formation Prior to Implantation of Bone Marrow-Derived Mesenchymal Cells Improves Function in the Ischemic Myocardium.

Bone marrow-derived mesenchymal stem cells (BM-MSC) are reported to induce beneficial effects in the heart following ischemia, but a loss of these cells within hours of implantation could significantly diminish their long-term effect. We hypothesized that early coupling between BM-MSC and ischemic cardiomyocytes through gap junctions (GJ) may play an important role in stem cell survival and retention in the acute phase of myocardial ischemia. To determine the effect of GJ inhibition on murine BM-MSC in vivo, we induced ischemia in mice using 90 min left anterior descending coronary artery (LAD) occlusion followed by BM-MSC implantation and reperfusion. The inhibition of GJ coupling prior to BM-MSC implantation led to early improvement in cardiac function compared to mice in which GJ coupling was not inhibited. Our results with in vitro studies also demonstrated increased survival in BM-MSCs subjected to hypoxia after inhibition of GJ. While functional GJ are critical for the long-term integration of stem cells within the myocardium, early GJ communication may represent a novel paradigm whereby ischemic cardiomyocytes induce a "bystander effect" when coupled to newly transplanted BM-MSC and thus impair cell retention and survival.

A pumpless monolayer microfluidic device based on mesenchymal stem cell-conditioned medium promotes neonatal mouse in vitro spermatogenesis

BackgroundChildhood cancer treatment-induced gonadotoxicity causes permanent infertility/sub-infertility in nearly half of males. The current clinical and experimental approaches are limited to cryopreservation of prepubertal testicular strips and in vitro spermatogenesis which are inadequate to achieve the expanded spermatogonial stem/progenitor cells and spermatogenesis in vitro. Recently, we reported the supportive effect of bone marrow-derived mesenchymal cell co-culture which is inadequate after 14 days of culture in static conditions in prepubertal mouse testis due to lack of microvascular flow and diffusion. Therefore, we generated a novel, pumpless, single polydimethylsiloxane-layered testis-on-chip platform providing a continuous and stabilized microfluidic flow and real-time cellular paracrine contribution of allogeneic bone marrow-derived mesenchymal stem cells.MethodsWe aimed to evaluate the efficacy of this new setup in terms of self-renewal of stem/progenitor cells, spermatogenesis and structural and functional maturation of seminiferous tubules in vitro by measuring the number of undifferentiated and differentiating spermatogonia, spermatocytes, spermatids and tubular growth by histochemical, immunohistochemical, flow cytometric and chromatographic techniques.ResultsBone marrow-derived mesenchymal stem cell-based testis-on-chip platform supported the maintenance of SALL4(+) and PLZF(+) spermatogonial stem/progenitor cells, for 42 days. The new setup improved in vitro spermatogenesis in terms of c-Kit(+) differentiating spermatogonia, VASA(+) total germ cells, the meiotic cells including spermatocytes and spermatids and testicular maturation by increasing testosterone concentration and improved tubular growth for 42 days in comparison with hanging drop and non-mesenchymal stem cell control.ConclusionsFuture fertility preservation for male pediatric cancer survivors depends on the protection/expansion of spermatogonial stem/progenitor cell pool and induction of in vitro spermatogenesis. Our findings demonstrate that a novel bone marrow-derived mesenchymal stem cell-based microfluidic testis-on-chip device supporting the maintenance of stem cells and spermatogenesis in prepubertal mice in vitro. This new, cell therapy-based microfluidic platform may contribute to a safe, precision-based cell and tissue banking protocols for prepubertal fertility restoration in future.

Toxic Effects Associated With Neonicotinoid Exposure on Non-target Organisms: A Review

The neonicotinoid class of insecticide is nicotine-like neuro-toxicants used to control the pests of agriculture crops and ornamental plants. They act as selective agonists of acetylcholine receptors in the central nervous system of insect pests and work by disrupting their nerve impulses. Some of the properties of this class of insecticides are a long half-life in soil, low volatility, and higher water solubility, leading to their accumulation in soil, underground water, and water bodies due to surface runoff. This, in turn, results in exposure to many beneficial non-target aquatic and soil fauna such as arthropods, fish, birds, mammals, etc. Although it has a selective mode of action for insects, some in vivo and in vitro investigations have also shown toxicity in non-target invertebrates and vertebrates. Initially, neonicotinoid toxicity was observed in honey bees, which are essential pollinators of crops. Later, studies reported the accumulation of neonicotinoid residues leading to the mortality of aquatic fauna, including salt marsh and freshwater mosquitoes, brine shrimp, fleas, and crayfish. Imidacloprid exposure led to disruption of larval development in the Mayfly larvae. Also, earthworms that play a crucial role in enhancing soil fertility were drastically affected by acetamiprid, clothianidin, imidacloprid, nitenpyram, and thiacloprid. Apart from these, toxicological impacts were also observed in vertebrates such as birds, where imidacloprid, clothianidin, acetamiprid, and thiacloprid caused reproductive, metabolic, and morphological alterations. Similarly, imidacloprid and acetamiprid caused gills, brain and liver dysfunction with embryo mortality. Even after the selective action of neonicotinoids, instances of mammalian toxicity were also reported in many in vivo studies. DNA damage and liver dysfunctions due to imidacloprid in rabbits were observed in various studies. In a recent study, imidacloprid exposure led to DNA damage and oxidative stress in bone marrow-derived mesenchymal cells of buffalo. Also, many instances of neurotoxicity, reproductive toxicity, immunotoxicity, genotoxicity and cytotoxicity in mouse and rat models were observed due to different neonicotinoids. Many in vitro studies using mammalian cell lines have also established potential risks of neonicotinoid exposure. This review, therefore, is a compilation of various toxicity studies of different types of neonicotinoid pesticides in both nontarget invertebrates and vertebrates, including several kinds of toxicities caused in mammals with neonicotinoid exposure.

Formation of low-density electrospun fibrous network integrated mesenchymal stem cell sheet.

Cell sheets combined with electrospun fibrous mats represent an attractive approach for the repair and regeneration of injured tissues. However, the conventional dense electrospun mats as supportive substrates in forming "cell sheet on fiber mat" complexes suffer from problems of limiting the cellular function and eliciting a host response upon implantation. To give full play to the role of electrospun biomimicking fibers in forming quality cell sheets, this study proposed to develop a cell-fiber integrated sheet (CFIS) featuring a spatially homogeneous distribution of cells within the fiber structure by using a low-density fibrous network for cell sheet formation. A low-density electrospun polycaprolactone (PCL) fibrous network at a density of 103.8 ± 16.3 μg cm-2 was produced by controlling the fiber deposition for a short period of 1 min and subsequently transferred onto polydimethylsiloxane rings for facilitating cell sheet formation, in which rat bone marrow-derived mesenchymal cells were used. Using a dense electrospun PCL fibrous mat (481.5 ± 7.5 μg cm-2) as the control, it was found that cells on the low-density fibrous network (L-G) exhibited improved capacities in spreading, proliferation, stemness maintenance and matrix-remodeling during the process of CFIS formation. Structurally, the CFIS constructs revealed strong integration between the cells and the fibrous network, thus providing excellent cohesion and physical integrity to enable strengthening of the formed cell sheet. By contrast, the cell sheet formed on the dense fibrous mat (D-G) showed a two-layer (biphasic) structure due to the limitation of cellular invasion. Moreover, such engineered CFIS was identified with enhanced immunomodulatory effects by promoting LPS-stimulated macrophages towards an M2 phenotype in vitro. Our results suggest that the CFIS may be used as a native tissue equivalent "cell sheet" for improving the efficacy of the tissue engineering approach for the repair and regeneration of impaired tissues.

Ex vivo study of detergent-assisted intraosseous bone wash treatment of osteonecrosis.

Avascular necrosis (AVN) involves ischemic cell death of the bone. AVN leaves an abundance of necrotic lipids and debris in the bone marrow, which instigates inflammatory bone repair. Consequently, the necrotic bone microenvironment stimulates excessive bone resorption, leading to joint deformities and osteoarthritis. Here, we performed a detergent-assisted bone wash using poloxamer 407 (P407) to clean the necrotic bone environment by removing lipids and necrotic debris. The new concept was tested using an established ex vivo AVN model of porcine cadaver humeral heads. The P407 wash was performed using P407 solution and followed with saline via two intraosseous needles. Visual inspection and image analyses of average pixel light intensity showed that the P407 wash produced a better-cleaned bone than the saline wash. Analyses of the collected bone wash solution showed a two-fold increase in triglycerides (101 vs. 53 mmol/head, p = 0.006) and a 10-fold increase in the dry weight of the removed debris (1.34 vs. 0.13 g/head, p = 0.02) with the P407 wash compared to saline. The histological evaluation showed significantly decreased Oil-Red-O (fats) staining in the P407-washed bone compared with the saline-washed bone. The in vitro assays of Alizarin red and qPCR showed the P407 wash neither altered the osteogenic behaviors of porcine bone marrow-derived mesenchymal cells (pBMMCs) nor raised inflammatory responses of porcine bone marrow-derived macrophages (pBMMs). In conclusion, detergent-assisted bone wash using P407 produced a better removal of nonsoluble debris from the bone marrow space than the saline wash without causing changes to osteogenesis or inflammatory reactions.

Binucleated human bone marrow-derived mesenchymal cells can be formed during neural-like differentiation with independence of any cell fusion events

Although it has been reported that bone marrow-derived cells (BMDCs) can transdifferentiate into neural cells, the findings are considered unlikely. It has been argued that the rapid neural transdifferentiation of BMDCs reported in culture studies is actually due to cytotoxic changes induced by the media. While transplantation studies indicated that BMDCs can form new neurons, it remains unclear whether the underlying mechanism is transdifferentiation or BMDCs-derived cell fusion with the existing neuronal cells. Cell fusion has been put forward to explain the presence of gene-marked binucleated neurons after gene-marked BMDCs transplantation. In the present study, we demostrated that human BMDCs can rapidly adopt a neural-like morphology through active neurite extension and binucleated human BMDCs can form with independence of any cell fusion events. We also showed that BMDCs neural-like differentiation involves the formation of intermediate cells which can then redifferentiate into neural-like cells, redifferentiate back to the mesenchymal fate or even repeatedly switch lineages without cell division. Furthermore, we have discovered that nuclei from intermediate cells rapidly move within the cell, adopting different morphologies and even forming binucleated cells. Therefore, our results provide a stronger basis for rejecting the idea that BMDCs neural transdifferentiation is merely an artefact.

Dysregulated transforming growth factor-beta mediates early bone marrow dysfunction in diabetes

Diabetes affects select organs such as the eyes, kidney, heart, and brain. Our recent studies show that diabetes also enhances adipogenesis in the bone marrow and reduces the number of marrow-resident vascular regenerative stem cells. In the current study, we have performed a detailed spatio-temporal examination to identify the early changes that are induced by diabetes in the bone marrow. Here we show that short-term diabetes causes structural and molecular changes in the marrow, including enhanced adipogenesis in tibiae of mice, prior to stem cell depletion. This enhanced adipogenesis was associated with suppressed transforming growth factor-beta (TGFB) signaling. Using human bone marrow-derived mesenchymal progenitor cells, we show that TGFB pathway suppresses adipogenic differentiation through TGFB-activated kinase 1 (TAK1). These findings may inform the development of novel therapeutic targets for patients with diabetes to restore regenerative stem cell function.

PAP Polypeptide Promotes Osteogenesis in Jaw Bone Defect Repair by Inhibiting Inflammatory Reactions.

Jaw defects are common in oral and maxillofacial diseases and require surgical repair in extreme cases. Given the limitations in availability and efficacy of autologous bone grafts or allografts, great effort has been made in finding suitable, biocompatible, and effective artificial bone materials. Considering the key role of inflammation in bone resorption, we sought to identify a polypeptide with anti-inflammatory and bone-promoting effects. Rat bone marrow-derived mesenchymal cells (BMSCs) were treated with lipopolysaccharide (LPS) to induce an inflammatory environment, and 1,538 differentially abundant polypeptides were identified using mass spectrometry. Based on mass spectrometry signal intensity, multiple of difference, and structural stability, PAP was screened out as the polypeptide with the lowest abundance in the inflammatory condition. PAP showed no cytotoxicity to BMSCs with increasing concentrations. PAP (10 μM) also increased alkaline phosphatase activity and mRNA expression of Ocn, Bmp2, and Runx2 in a concentration-dependent manner, which confirmed that it can promote osteogenic induction of rat BMSCs. Moreover, PAP reduced LPS-induced expression of inflammatory cytokines (TNF-α, IL-1β, IL-6) and reactive oxygen species and inhibited polarization of RAW 264.7 macrophages to the inflammatory type. Finally, a skull defect mouse model was established, and mice were injected with LPS and/or PAP. Micro-CT, histological analysis, and immunohistochemical staining showed that PAP significantly reduced the number of LPS-induced bone resorption pits and maintained bone integrity. Overall, the polypeptide PAP screened using LPS stimulation of BMSCs is not cytotoxic and can inhibit the inflammatory reaction process to promote osteogenesis. This study thus provides a basis for development of PAP as a new osteogenic material in the repair of jaw defects.

Simulated confluence on micropatterned substrates correlates responses regulating cellular differentiation.

While cells are known to behave differently based on the size of micropatterned islands, and this behavior is thought to be related to cell size and cell-cell contacts, the exact threshold for this difference between small and large islands is unknown. Furthermore, while cell size and cell-cell contacts can be easily manipulated on small islands, they are harder to measure and continually monitor on larger islands. To investigate this size threshold, and to explore cell size, cell-cell contacts, and differentiation, we use a previously established simulation to plan experiments and explain results that we could not explain from experiments alone. We use five seeding densities covering three orders of magnitude over 25-500 µm diameter islands to examine markers of proliferation and differentiation in bone marrow-derived mesenchymal cells (cell line). We show that osteogenic markers are most accurately described as a function of confluence for larger islands, but a function of time for smaller islands. We further show, using results of the simulation, that cell size and cell-cell contacts are also related to confluence on larger islands, but only cell-cell contacts are related to confluence on small islands. This study uses simulations to explain experimental results that could not be explained from experiments alone. Together, the simulations and experiments in this study show different differentiation patterns on large and small islands, and this simulation may be useful in planning future studies related to this study.

Safety of Intravenous Autologous Bone Marrow-Derived Mesenchymal Cell Transplantation in 5 Patients With Reduced Left Ventricular Ejection Fraction.

Background: Although intracardiac injection or intracoronary delivery of mesenchymal stem cells (MSCs) has been reported, there have been few studies on the intravenous injection of MSCs, particularly in Japan.Methods and Results: Five patients with left ventricular ejection fraction (LVEF) ≤45% received 1.0×108 MSCs intravenously. The procedure did not induce significant changes in vital signs. One patient had an elevated body temperature after 1 day, but recovered spontaneously. Laboratory tests remained normal for 1 month after cell delivery. Computed tomography was performed after 1–2 years, and there was no evidence of malignancy.Conclusions: In this pilot study of patients with reduced LVEF, intravenous MSC delivery had no adverse effects.

Cellular therapies in kidney transplantation.

Current immunosuppressive regimens used in kidney transplantation are sometimes ineffective and carry significant risks of morbidity and mortality. Cellular therapies are a promising alternative to prolong graft survival while minimizing treatment toxicity. We review the recently published breakthrough studies using cell therapies in kidney transplantation. The reviewed phase I and II trials showed that cell therapies are feasible and safe in kidney transplantation, sometimes associated with less infectious complications than traditional regimens. Regulatory T cells and macrophages were added to the induction regimen, allowing for lower immunosuppressive drug doses without higher rejection risk. Regulatory T cells are also a treatment for subclinical rejection on the 6 months biopsy. Other strategies, like bone marrow-derived mesenchymal cells, genetically modified regulatory T cells, and chimerism-based tolerance are also really promising. In addition, to improve graft tolerance, cell therapy could be used to prevent or treat viral infection after transplantation. Emerging data underline that cell therapy is a feasible and safe treatment in kidney transplantation. Although the evidence points to a benefit for transplant recipients, studies with standardized protocols, representative control groups, and longer follow-up are needed to answer the question definitively and guide future research.

Extracellular vesicles containing miR-146a-5p secreted by bone marrow mesenchymal cells activate hepatocytic progenitors in regenerating rat livers

BackgroundSmall hepatocyte-like progenitor cells (SHPCs) appear to form transient clusters in rat livers treated with retrorsine (Ret) and 70% partial hepatectomy (PH). We previously reported that the expansion of SHPCs was amplified in Ret/PH-treated rat livers transplanted with Thy1+ cells derived from d-galactosamine-treated injured livers. Extracellular vesicles (EVs) produced by hepatic Thy1+ donor cells activated SHPCs via interleukin (IL)-17 receptor B signaling. As bone marrow-derived mesenchymal cells (BM-MCs) also express Thy1, we aimed to determine whether BM-MCs could also promote the growth of SHPCs.MethodsBM-MCs were isolated from dipeptidyl-peptidase IV (DPPIV)-positive rats. BM-MCs or BM-MC-derived EVs were administered to DPPIV-negative Ret/PH rat livers, and the growth and the characteristics of SHPC clusters were evaluated 14 days post-treatment. miRNA microarrays and cytokine arrays examined soluble factors within EVs. Small hepatocytes (SHs) isolated from an adult rat liver were used to identify factors enhancing hepatocytic progenitor cells growth.ResultsThe recipient’s livers were enlarged at 2 weeks post-BM-MC transplantation. The number and the size of SHPCs increased remarkably in livers transplanted with BM-MCs. BM-MC-derived EVs also stimulated SHPC growth. Comprehensive analyses revealed that BM-MC-derived EVs contained miR-146a-5p, interleukin-6, and stem cell factor, which could enhance SHs’ proliferation. Administration of EVs derived from the miR-146a-5p-transfected BM-MCs to Ret/PH rat livers remarkably enhanced the expansion of SHPCs.ConclusionsmiR-146a-5p involved in EVs produced by BM-MCs may play a major role in accelerating liver regeneration by activating the intrinsic hepatocytic progenitor cells.

Neonatal hyperoxia impairs adipogenesis of bone marrow-derived mesenchymal stem cells and fat accumulation in adult mice

Preterm birth is a risk factor for growth failure and development of respiratory disease in children and young adults. Their early exposure to oxygen may contribute to lung disease because adult mice exposed to hyperoxia as neonates display reduced lung function, changes in the host response to respiratory viral infections, and develop pulmonary hypertension and heart failure that shortens their lifespan. Here, we provide new evidence that neonatal hyperoxia also impairs growth by inhibiting fat accumulation. Failure to accumulate fat may reflect a systemic defect in adipogenic potential of stem cells because bone marrow-derived mesenchymal cells (BMSCs) isolated from the mice grew slower and were more oxidized compared to controls. They also displayed reduced capacity to accumulate lipid and differentiate into adipocytes. BMSCs from adult mice exposed to neonatal hyperoxia express lower levels of peroxisome proliferator-activated receptor gamma (PPARγ), a transcription factor that drives adipocyte differentiation. The defect in adipogenesis was rescued by expressing PPARγ in these cells. These findings reveal early life exposure to high levels of oxygen may suppresses fat accumulation and impair adipogenic differentiation upstream of PPARγ signaling, thus potentially contributing to growth failure seen in people born preterm.

Biomolecule from Trigonella stellata from Saudi Flora to Suppress Osteoporosis via Osteostromal Regulations.

Trigonella stellata has used in folk medicine as palatable and nutraceutical herb. It also regulates hypocholesterolemia, hypoglycemia, and has showed anti-inflammatory activities as well as antioxidants efficacy. Osteoporosis is a one of bone metabolic disorders and is continuously increasing worldwide. In the present study, caffeic acid was isolated from Trigonella stellata and identified using 1 D- and 2 D-NMR spectroscopic data. Caffeic acid was investigated on osteoblast and osteoclast in vitro using mice bone marrow-derived mesenchymal cells. Caffeic acid played reciprocal proliferation between osteoblast and osteoclast cells and accelerated the bone mineralization. It was confirmed by cytotoxicity, alkaline phosphatase (ALP), alizarin red S (ARS), and Tartrate resistant acid phosphatase (TRAP) assay. Caffeic acid regulated the osteogenic marker and upregulated the osteopontin, osteocalcin, and bone morphogenic proteins (BMP). Quantitative real time PCR and Western blot were used to quantify the mRNA and protein markers. It also regulated the matrix metalloprotease-2 (MMP-2) and cathepsin-K proteolytic markers in osteoclast cells. In addition, caffeic acid inhibited bone resorption in osteoclast cells. On the other hand, it upregulate osteoblast differentiation through stimulation of extracellular calcium concentrations osteoblast differentiation, respectively. The results also were confirmed through in silico docking of caffeic acid against cathepsin-B and cathepsin-K markers. These findings revealed that caffeic acid has a potential role in bone-metabolic disorder through its multifaceted effects on osteoblast and osteoclast regulations and controls osteoporosis.

Abstract 15904: Echogenic Immunoliposome-targeted Mesenchymal Stem Cells for Imaging and Treatment of Atheroma

Background: Vascular stem cell dead impairs the regenerating capacity of the arterial wall with atherosclerosis. A patented technology has been developed to employ biofunctional echogenic immunoliposomes (BF-ELIP) for enhancing entry of stem cells to atherosclerotic lesions. Objective: Determine whethere ultrasound-enhanced delivery of BF-ELIP-targeted stem cells visualize and treat atheroma simultaneously. Methods and Results: BF-ELIP were prepared by conjugating thiolated antibodies specific for the mesenchymal marker CD146 and murine ICAM-1 to phosphatidyl-ethanolamine liposomes through a thioether linkage. CD146 + bone marrow-derived mesenchymal cells (BM-MSC) were prepared from green fluorescence protein (GFP)-transgenic mice. By fluorescence microscopy, BF-ELIP targeted CD146 + cells were clearly visualized being adherent on cultured human endothelial cells that expressed ICAM-1 induced by tumor necrosis factor alpha (TNF-α) (Fig. 1a). BM-MSC/BF-ELIP mixtures were intravenously injected into ApoE-/- mice (n=9). Ultrasound illustrated intense echo signals of BF-ELIP in the aortic arch 15 min after injection of the BM-MSC/BF-ELIP preparation into ApoE-knockout mice (Fig. 1b). Histopathology of the aorta analyzed by H&E and Oil-Red O staining of aortic sections showed that the BF-ELIP targeted MSC delivery reduced plaque sizes, inflammation and lipid loading. Neither normal wild type C57BL/6J nor ApoE-knockout mice without injection showed the intensified echo signals and histological changes in their aortic arch regions. Conclusions: BF-ELIP enhancement of vascular stem cell adherence to inflammatory endothelium facilitates vulnerable plaque detection and targeted stem cell therapy for atheroma. This proof of concept study illustrates on the development of ultrasound-enhanced, BF-ELIP-mediated stem cell delivery and therapeutic approach to stabilize atheroma.

Coordinated Regulation of Mesenchymal Stem Cell Migration by Various Chemotactic Stimuli.

Endogenous bone marrow-derived mesenchymal stem cells are mobilized to peripheral blood and injured tissues in response to changes in the expression of various growth factors and cytokines in the injured tissues, including substance P (SP), transforming growth factor-beta (TGF-β), and stromal cell-derived factor-1 (SDF-1). SP, TGF-β, and SDF-1 are all known to induce the migration of bone marrow-derived mesenchymal stem cells (BM-MSCs). However, it is not yet clear how these stimuli influence or interact with each other during BM-MSC mobilization. This study used mouse bone marrow-derived mesenchymal stem cell-like ST2 cells and human BM-MSCs to evaluate whether SP, TGF-β, and SDF-1 mutually regulate their respective effects on the mobilization of BM-MSCs. SP pretreatment of ST2 and BM-MSCs impaired their response to TGF-β while the introduction of SP receptor antagonist restored the mobilization of ST2 and BM-MSCs in response to TGF-β. TGF-β pretreatment did not affect the migration of ST2 and BM-MSCs in response to SP, but downregulated their migration in response to SDF-1. SP pretreatment modulated the activation of TGF-β noncanonical pathways in ST2 cells and BM-MSCs, but not canonical pathways. These results suggest that the migration of mesenchymal stem cells is regulated by complex functional interactions between SP, TGF-β, and SDF-1. Thus, understanding the complex functional interactions of these chemotactic stimuli would contribute to ensuring the development of safe and effective combination treatments for the mobilization of BM-MSCs.

Sox10 Functions as an Inducer of the Direct Conversion of Keratinocytes Into Neural Crest Cells.

Neural crest cells (NCCs) are highly migratory multipotent cells that play critical roles in embryogenesis. The generation of NCCs is controlled by various transcription factors (TFs) that are regulated by each other and combine to form a regulatory network. We previously reported that the conversion of mouse fibroblasts into NCCs was achieved by the overexpression of only one TF, Sox10; therefore, Sox10 may be a powerful inducer of the conversion of NCCs. We herein investigated whether Sox10 functions in the direct conversion of other somatic cells into NCCs. Sox10 directly converted bone marrow-derived mesenchymal cells, but not keratinocytes, into P75+ NCCs. However, by the co-expression of four TFs (Snail1, Snail2, Twist1, and Tcfap2a) that are involved in NCC generation, but unable convert cells into NCCs, Sox10 converted keratinocytes into P75+ NCCs. P75+ NCCs mainly differentiated into glial cells, and to a lesser extent into neuronal cells. On the other hand, when Sox10 was expressed after the four TF expression, which mimicked the expression order in in vivo NCC generation, it converted keratinocytes into multipotent NCCs. These results demonstrate that Sox10 functions as an inducer of direct conversion into NCCs in cooperation with the TFs involved in NCC generation. The sequence of expression of the inducer and cooperative factors is important for the conversion of somatic cells into bona fide target cells.