Undifferentiated Mesenchymal Stromal Cells Research Articles

Cigarette smoke and nicotine effect on human mesenchymal stromal cell wound healing and osteogenic differentiation capacity.

Mesenchymal stromal cells (MSCs) play a crucial role in promoting tissue regeneration and healing, particularly in bone tissue. Both smoking and nicotine use are known to delay and inhibit the healing process in patients. This study aims at delineating these cellular effects by comparing the impact of nicotine alone to cigarette smoke with equivalent nicotine content, and shedding light on potential differences in the healing process. We examined how cigarette smoke and nicotine affect the migration, proliferation, and osteogenic differentiation of human patient-derived MSCs in vitro, as well as the secretion of cytokines IL-6 and IL-8. We measured nicotine concentration of the cigarette smoke extract (CSE) to clarify the role of the nicotine in the effect of the cigarette smoke. MSCs exposed to nicotine-concentration-standardized CSE exhibited impaired wound healing capability, and at high concentrations, increased cell death. At lower concentrations, CSE dose-dependently impaired migration, proliferation, and osteogenic differentiation, and increased IL-8 secretion. Nicotine impaired proliferation and decreased PINP secretion. While there was a trend for elevated IL-6 levels by nicotine in undifferentiated MSCs, these changes were not statistically significant. Exposure of MSCs to equivalent concentrations of nicotine consistently elicited stronger responses by CSE and had a more pronounced effect on all studied parameters. Our results suggest that the direct effect of cigarette smoke on MSCs contributes to impaired MSC function, that adds to the nicotine effects. Cigarette smoke extract reduced the migration, proliferation, and osteogenic differentiation in MSCs in vitro, while nicotine alone reduced proliferation. Cigarette smoke impairs the osteogenic and regenerative ability of MSCs in a direct cytotoxic manner. Cytotoxic effect of nicotine alone impairs regenerative ability of MSCs, but it only partly explains cytotoxic effects of cigarette smoke. Direct effect of cigarette smoke, and partly nicotine, on MSCs could contribute to the smoking-related negative impact on long-term bone health, especially in bone healing.

Glutamine Addiction of Multiple Myeloma Cells Reprograms Mesenchymal Stromal Cell Enzymes and Transporters Towards a Pro-Tumor Phenotype

Metabolic alterations in cancer are not only functional to ensure malignant cell growth but also shape a pro-tumor behavior of normal cell populations in the tumor microenvironment. Multiple myeloma (MM) is the only human cancer that is both glutamine-addicted and glutamine-auxotroph, a feature that renders MM growth completely dependent upon extracellular glutamine availability. Indeed, plasma cells from most MM patients do not express Glutamine Synthetase (GS), the only enzyme able to synthetize glutamine from glutamate and ammonium, while express high levels of Glutaminase (GLS), which catalyze the first step of glutaminolysis by deamidating glutamine into glutamate and ammonium. As a consequence, the bone marrow (BM) plasma of MM patients has low-glutamine/high glutamate levels compared to patients with smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS), as firstly demonstrated by our group. This particular metabolic microenvironment induces GS expression in mesenchymal stromal cells (MSCs) impairing osteoblast (OB) differentiation of MSCs, thus favoring MM bone lesions typical of active MM. The impact of these metabolic alterations on other BM cell populations is much less defined and it has been investigated in this study. For instance, although MM BM displays an oversized adipocyte population, which sustain MM growth by providing free fatty acids, the effects of MM metabolism on MSCs and their adipocyte differentiation remain to be characterized. First we show that 13C 5-Gln isotopomer distribution revealed that almost 50% of total glutamate in human myeloma cell lines (HMLCs) directly derives from glutamine deamidation by GLS. Furthermore, in standard culture conditions, HMLCs secreted glutamate likely through the SLC7A11 transporter, whose activity was higher in HMLCs than in MSCs. On the other hand, HMCLs were not able to take up glutamate from the extracellular space, while human primary MSCs isolated from healthy donors displayed high activity of the sodium-dependent glutamate transporters of the EAAT family. Moreover, glutamate uptake was higher in undifferentiated MSCs than in MSC incubated for 14 days in osteogenic medium (10 -8 M dexamethasone and 50 µg/ml ascorbic acid). Consistently, a public transcriptional profile of MSCs and OBs obtained from bone biopsies of healthy donors (n=7) or MM patients (n=16) revealed that the expression of the inward Glu transporter EAAT3 is higher in MSCs compared to OBs. In glutamine-free conditions, MSCs produced and secreted glutamine, a process boosted by extracellular glutamate in a dose-dependent manner. Glutamine secretion was hindered by either l-methionine sulfoximine (MSO), an irreversible inhibitor of GS, or D-aspartate (D-Asp), a high-affinity inhibitor of EAAT3 that hinders glutamate uptake. In a co-culture system, HMCLs induced the expression of SNAT5 glutamine efflux transporter in MSCs. Consistently, upon glutamine withdrawal, primary undifferentiated MSCs from healthy donors sustained HMCLs growth, while this nutritional support was markedly impaired by either inhibition or silencing of GS and EAAT3, with a substantial decrease in MM cell viability. Lastly, primary human MSCs were incubated under adipogenic conditions (0.5 mM 3-Isobutyl-1-methylxanthine, 5 µM indomethacin, 50 µM dexamethasone and 10 mg/ml human insulin) in the presence or in the absence of glutamine to mimic, respectively, normal or MM BM microenvironment. Glutamine deprivation increased lipogenesis, assessed with Oil Red O staining, as well as the expression of the adipocyte markers PPARG, LEP, and ADIPOQ. These data point to a MM-driven metabolic pro-tumor BM niche in which MSCs feed glutamine-addicted MM cells, and MSC differentiation is skewed from osteogenesis to adipogenesis. Several steps of these deranged pathways are amenable to pharmacological inhibition, pointing to possible novel therapeutic approaches to counteract MM growth and its effects on the BM niche.

MicroRNAs as prognostic markers for chondrogenic differentiation potential of equine mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) are a promising cell source for cartilage tissue regeneration in animals and humans but with large inter-donor variation in their in vitro chondrogenic differentiation potential. Underlying molecular mechanisms responsible for culture-expanded MSC heterogeneity remains poorly understood. In this study, we sought to identify variations in miRNA signatures associated with cultured equine MSC chondrogenic differentiation potential from different donors. Neocartilage tissue generated from equine cord blood-derived MSCs (CB-MSCs) was categorized as having either high or low chondrogenic potential based on their histological appearance and quantification of glycosaminoglycan deposition. Using next-generation sequencing, we identified 30 differentially expressed miRNAs amongst undifferentiated MSC cultures that corresponded with their chondrogenic potential. Of note, MSCs with low chondrogenic potential upregulated miR-146a and miR-487b-3p, which was also observed by qRT-PCR. Our findings suggest that miRNA profiling of equine MSC cultures may have prognostic value in selecting MSC donors with regards to their chondrogenic differentiation potential.

Micromolding-based encapsulation of mesenchymal stromal cells in alginate for intraarticular injection in osteoarthritis.

Osteoarthritis (OA) is an inflammatory joint disease that affects cartilage, subchondral bone, and joint tissues. Undifferentiated Mesenchymal Stromal Cells are a promising therapeutic option for OA due to their ability to release anti-inflammatory, immuno-modulatory, and pro-regenerative factors. They can be embedded in hydrogels to prevent their tissue engraftment and subsequent differentiation. In this study, human adipose stromal cells are successfully encapsulated in alginate microgels via a micromolding method. Microencapsulated cells retain their in vitro metabolic activity and bioactivity and can sense and respond to inflammatory stimuli, including synovial fluids from OA patients. After intra-articular injection in a rabbit model of post-traumatic OA, a single dose of microencapsulated human cells exhibit properties matching those of non-encapsulated cells. At 6 and 12 weeks post-injection, we evidenced a tendency toward a decreased OA severity, an increased expression of aggrecan, and a reduced expression of aggrecanase-generated catabolic neoepitope. Thus, these findings establish the feasibility, safety, and efficacy of injecting cells encapsulated in microgels, opening the door to a long-term follow-up in canine OA patients.

Minimally Invasive Management of a Rare Giant Cell Tumor of Talus: A Case Report and Literature Review

Category:Ankle; Hindfoot; OtherIntroduction/Purpose:Giant cell tumor (GCT) of small bones of hand and feet is rare. A benign but locally aggressive neoplasm with a tendency for local recurrence with undifferentiated mesenchymal stromal cells and the presence of abundant, multinucleated giant cells. It occurs in a slightly younger age group with usually an expansile and eccentric epiphyseo-metaphyseal region lytic lesion. A 22 yrs. old patient, presenting as lytic expansile lesion of talus, suspected Giant cell tumor (GCT) of talus preoperatively and intraoperatively and proven subsequently, is presented along with review of literature. This patient is the young and nearly 24th case reported in the literature for GCT of talus.Methods:Author reviewed the literature for the GCT of talus from 1964 to till 2021. The author reported a case of left ankle joint pain and swelling for 6 months in a 22 yrs. old male, which turns out to be aneurysmal bone cyst of talus on radiographs, Computed tomography and aspiration cytology. Campanacci grade II lesion was planned for the curettage and autologous bone grafting.Results:Retrospectively, the intraoperative biopsy confirmed the lesion as giant cell tumor with aneurysmal bone cyst. After 4 years the lesion showed the near complete resolution on radiographs and CT scan. However, because of the historical recurrence and metastasis reports of GCT, author is continuously monitoring patient's ankle with yearly follow up. Author reviwed the literarue for the GCT of talus from 1964 to till 2021. Author found total of 23 reported cases for GCT of talus in literature, out of which 2 cases showed recurrence and distant metastasis to chest which is 8.69 percent of total cases.Conclusion:Author concludes that the Campanacci grade I and II can be managed with mini-open lesional curettage and autologous bone grafting while grade III and IV needed osteotomy, en-bloc excisions and tibio-calcaneal fusion or below knee amputation in recurrent or aggressive lesions. As author found 8.69 percent cases of distant pulmonary metastasis out of total reported cases, he recommends yearly follow up of the lesions and look for distant metastasis in suspected cases.

The therapeutic potential ofadipose tissue-derived mesenchymal stromal cellsin thetreatmentof busulfan-induced azoospermic mice.

The generation of germ cells from mesenchymal stromal cells (MSCs) provides a valuable in vitro platform for infertility modeling. The establishment of these cells is a new approach for assisted reproductive technology (ART) to help infertile patients who lack functional gametes. Human adipose-derived MSCs were isolated and then characterized for multipotency by flow cytometry, differentiation capacity, and cytogenetic assays. These cells were used in a male germ cell differentiation study. The expression of male germ cell markers was evaluated at day 21 of differentiation using an immunofluorescence assay, flow cytometry, and RT-qPCR. Undifferentiated MSCs were used for transplantation in busulfan-induced azoospermic mice. In this study, MSCs were successfully isolated from human adipose tissues which were positive for cell markers such as CD90, CD105, CD73, and CD29 but negative for CD34 and CD45. The results of flow cytometry, immunocytochemistry, and RT-qPCR analysis at day 21 of differentiation showed that the undifferentiated adipose-derived MSCs are able to differentiate into male germ cells. Additionally, transplantation of undifferentiated MSCs in busulfan-induced azoospermic mice caused spermatogenesis recovery in the majority of seminiferous tubules. In this study, we showed that differentiation of human adipose-derived MSCs into male germ cells is a useful tool for in vitro study of human germ cell development. Our results demonstrated that cell therapy with adipose-derived MSCs could help the repair of pathological changes in testicular seminiferous tubules. Therefore, it may have a clinical application for the treatment of azoospermia in infertile patients.

Insulin-producing cells from mesenchymal stromal cells: Protection against cognitive impairment in diabetic rats depends upon implant site

Diabetes mellitus (DM) is a serious public health problem and can cause long-term damage to the brain, resulting in cognitive impairment in these patients. Insulin therapy for type 1 DM (DM1) can achieve overall blood glucose control, but glycemic variations can occur during injection intervals, which may contribute to some complications. Among the additional therapies available for DM1 treatment is the implantation of insulin-producing cells (IPCs) to attenuate hyperglycemia and even reverse diabetes. Here, we studied the strategy of implanting IPCs obtained from mesenchymal stromal cells (MSCs) from adipose tissue, comparing two different IPC implant sites, subcapsular renal (SR) and subcutaneous (SC), to investigate their putative protection against hippocampal damage, induced by STZ, in a rat DM1 model. Both implants improved hyperglycemia and reduced the serum content of advanced-glycated end products in diabetic rats, but serum insulin was not observed in the SC group. The SC-implanted group demonstrated ameliorated cognitive impairment (evaluated by novel object recognition) and modulation of hippocampal astroglial reactivity (evaluated by S100B and GFAP). Using GFP+ cell implants, the survival of cells at the implant sites was confirmed, as well as their migration to the pancreas and hippocampus. The presence of undifferentiated MSCs in our IPC preparation may explain the peripheral reduction in AGEs and subsequent cognitive impairment recovery, mediated by autophagic depuration and immunomodulation at the hippocampus, respectively. Together, these data reinforce the importance of MSCs for use in neuroprotective strategies, and highlight the logistic importance of the subcutaneous route for their administration.

Seeding decellularized nerve allografts with adipose-derived mesenchymal stromal cells: An in vitro analysis of the gene expression and growth factors produced

Mesenchymal stromal cells (MSCs) secrete many soluble growth factors and have previously been shown to stimulate nerve regeneration. MSC-seeded processed nerve allografts could potentially be a promising method for large segmental motor nerve injuries. Further progress in our understanding of how the functions of MSCs can be leveraged for peripheral nerve repair is required before making clinical translation. The present study, therefore, investigated whether interactions of adipose-derived MSCs with decellularized nerve allografts can improve gene and protein expression of growth factors that may support nerve regeneration. Human nerve allografts (n = 30) were decellularized and seeded with undifferentiated human adipose-derived MSCs. Subsequently, the MSCs and MSC-seeded grafts were isolated on days 3, 7, 14, and 21 in culture for RNA expression analysis by qRT-PCR. Evaluated genes included NGF, BDNF, PTN, GAP43, MBP, PMP22, VEGF, and CD31. Growth factor production was evaluated and quantified using enzyme-linked immunosorbent assay (ELISA). On day 21, semi-quantitative RT-PCR analysis showed that adherence of MSCs to nerve allografts significantly enhances mRNA expression of neurotrophic, angiogenic, endothelial, and myelination markers (e.g., BDNF, VEGF, CD31, and MBP). ELISA results revealed an upregulation of BDNF and reduction of both VEGF and NGF protein levels. This study demonstrates that seeding of undifferentiated adipose-derived MSCs onto processed nerve allografts permits the secretion of neurotrophic and angiogenic factors that can stimulate nerve regeneration. These favorable molecular changes suggest that MSC supplementation of nerve allografts may have potential in improving nerve regeneration.

Injectable osteogenic microtissues containing mesenchymal stromal cells conformally fill and repair critical-size defects

Repair of complex fractures with bone loss requires a potent, space-filling intervention to promote regeneration of bone. We present a biomaterials-based strategy combining mesenchymal stromal cells (MSC) with a chitosan-collagen matrix to form modular microtissues designed for delivery through a needle to conformally fill cavital defects. Implantation of microtissues into a calvarial defect in the mouse showed that osteogenically pre-differentiated MSC resulted in complete bridging of the cavity, while undifferentiated MSC produced mineralized tissue only in apposition to native bone. Decreasing the implant volume reduced bone regeneration, while increasing the MSC concentration also attenuated bone formation, suggesting that the cell-matrix ratio is important in achieving a robust response. Conformal filling of the defect with microtissues in a carrier gel resulted in complete healing. Taken together, these results show that modular microtissues can be used to augment the differentiated function of MSC and provide an extracellular environment that potentiates bone repair.

Paracrine Effects of Mesenchymal Stromal Cells Cultured in Three-Dimensional Settings on Tissue Repair.

Mesenchymal stromal cells (MSCs) are a promising cell source for promoting tissue repair, due to their ability to release growth, angiogenic, and immunomodulatory factors. However, when injected as a suspension, these cells suffer from poor survival and localization, and suboptimal release of paracrine factors. While there have been attempts to overcome these limitations by modifying MSCs themselves, a more versatile solution is to grow them in three dimensions, as aggregates or embedded into biomaterials. Here we review the mechanisms by which 3D culture can influence the regenerative capacity of undifferentiated MSCs, focusing on recent examples from the literature. We further discuss how knowledge of these mechanisms can lead to strategic design of MSC therapies that overcome some of the challenges to their effective translation.

Application of tissue-engineered bone grafts for alveolar cleft osteoplasty in a rodent model.

The clinical standard for alveolar cleft osteoplasty is augmentation with autologous bone being available in limited amounts and might be associated with donor site morbidity. The aim of the present study was the creation of tissue-engineered bone grafts and their in vivo evaluation regarding their potential to promote osteogenesis in an alveolar cleft model. Artificial bone defects with a diameter of 3.3mm were created surgically in the palate of 84 adult Lewis rats. Four experimental groups (n=21) were examined: bovine hydroxyl apatite/collagen (bHA) without cells, bHA with undifferentiated mesenchymal stromal cells (MSC), bHA with osteogenically differentiated MSC. In a control group, the defect remained empty. After 6, 9 and 12weeks, the remaining defect volume was assessed by cone beam computed tomography. Histologically, the remaining defect width and percentage of bone formation was quantified. After 12weeks, the remaining defect width was 60.1% for bHA, 74.7% for bHA with undifferentiated MSC and 81.8% for bHA with osteogenically differentiated MSC. For the control group, the remaining defect width measured 46.2% which was a statistically significant difference (p<0.001). The study design was suitable to evaluate tissue-engineered bone grafts prior to a clinical application. In this experimental set-up with the described maxillary defect, no promoting influence on bone formation of bone grafts containing bHA could be confirmed. The creation of a sufficient tissue-engineered bone graft for alveolar cleft osteoplasty could preserve patients from donor site morbidity.

Identification of Factors Produced and Secreted by Mesenchymal Stromal Cells with the SILAC Method.

Mesenchymal stromal cells (MSCs) secrete a large variety of proteins and factors, which shape the secretome. These proteins participate in multiple cellular functions, including the promotion of regenerative processes in the damaged tissue. Secretomes derived from either undifferentiated MSCs or these cells undergoing osteogenic, chondrogenic, or adipogenic differentiation have been characterized using different liquid chromatography tandem mass spectrometry (LC-MS/MS)-based quantitative proteomic approaches. In this chapter, we describe the use of the Stable Isotope Labeling by Amino Acids in Cell culture (SILAC) strategy for the identification and relative quantification of the mesenchymal stromal cell secretome, specifically during chondrogenesis.

Smooth Muscle-Like Cells Generated from Human Mesenchymal Stromal Cells Display Marker Gene Expression and Electrophysiological Competence Comparable to Bladder Smooth Muscle Cells.

The use of mesenchymal stromal cells (MSCs) differentiated toward a smooth muscle cell (SMC) phenotype may provide an alternative for investigators interested in regenerating urinary tract organs such as the bladder where autologous smooth muscle cells cannot be used or are unavailable. In this study we measured the effects of good manufacturing practice (GMP)-compliant expansion followed by myogenic differentiation of human MSCs on the expression of a range of contractile (from early to late) myogenic markers in relation to the electrophysiological parameters to assess the functional role of the differentiated MSCs and found that differentiation of MSCs associated with electrophysiological competence comparable to bladder SMCs. Within 1–2 weeks of myogenic differentiation, differentiating MSCs significantly expressed alpha smooth muscle actin (αSMA; ACTA2), transgelin (TAGLN), calponin (CNN1), and smooth muscle myosin heavy chain (SM-MHC; MYH11) according to qRT-PCR and/or immunofluorescence and Western blot. Voltage-gated Na+ current levels also increased within the same time period following myogenic differentiation. In contrast to undifferentiated MSCs, differentiated MSCs and bladder SMCs exhibited elevated cytosolic Ca2+ transients in response to K+-induced depolarization and contracted in response to K+ indicating functional maturation of differentiated MSCs. Depolarization was suppressed by Cd2+, an inhibitor of voltage-gated Ca2+-channels. The expression of Na+-channels was pharmacologically identified as the Nav1.4 subtype, while the K+ and Ca2+ ion channels were identified by gene expression of KCNMA1, CACNA1C and CACNA1H which encode for the large conductance Ca2+-activated K+ channel BKCa channels, Cav1.2 L-type Ca2+ channels and Cav3.2 T-type Ca2+ channels, respectively. This protocol may be used to differentiate adult MSCs into smooth muscle-like cells with an intermediate-to-late SMC contractile phenotype exhibiting voltage-gated ion channel activity comparable to bladder SMCs which may be important for urological regenerative medicine applications.

Mesenchymal Cell Associated Fibrosis in Experimental mplW515L Mouse Model of Myelofibrosis

Background: Bone marrow fibrosis caused by excessive collagen deposition represents a significant indicator of poor prognosis in myeloproliferative neoplasms (MPNs). Genetic driver mutations have been identified as JAK2, CALR and mpl, but the precise mechanism and cell type leading to fibrosis remains unanswered. TGFβ production by mutant hematopoietic cells is the primary factor responsible for fibrosis initiation. Although effective JAK2 inhibitor therapy has been adopted to manage disease symptoms and decrease abnormal hematopoietic cells, this pharmacological inhibitor does not reverse fibrosis. Our previous observations in patients with Large granular lymphocyte (LGL) leukemia indicate that mesenchymal-derived fibroblasts, distinguished by the capacity to undergo trilineage differentiation into osteoblasts, adipocytes and chondrocytes, are responsible for the fibrosis observed in these patients. In this study, we report the identification of a similar fibrosis-initiating mesenchymal cell population in a mouse model of MPN induced by transplantation of gfp-mplW515L retrovirus-expressing hematopoietic cells and indicate a mechanism to reverse the fibrotic phenotype.Methods: Primary mesenchymal stromal cells (MSCs) were isolated from mice with MPN caused by the transplantation of gfp-mplW515L -transduced bone marrow cells (MPN mice) and from control mice that received transplants with gfp-wild-type(wt)-mpl-expressing bone marrow. MSCs were harvested 17 days post-transplant when WBC counts exceeded at least >20,000 cells/μl. Fibrillar collagen I (detected with trichrome stain in bone marrow biopsies) and collagen III (detected with reticulin stain in bone marrow biopsies) were identified as the major proteins produced in the bone marrow and by cultured MSCs in MPN mice consistent with pathologic examination. The amount of collagen was quantified in MSCs grown under reduced oxygen in undifferentiating culture conditions. To determine pluripotency, cells were grown with differentiation-specific culture media and stained with Alizarin Red S (to detect osteoblasts) and Alcian blue (chondrocytes). Collagen production was determined by qRT-PCR and polychromatic immunofluorescence staining of three-dimensional cultures and TGFβ was measured by qRT-PCR and ELISA.Results: To isolate purified populations of MSCs, gfp-positive hematopoietic cells were removed and MSCs were maintained in long-term culture in the absence of malignant cells. MSCs were isolated in micro-cultures from gfp-wt-mpl (ie, control) and mplW515L transplanted mice. Using gfp-wt-mpl-derived MSCs, we first showed that the addition of TGFβ induced collagen I and III expression as expected from previous studies. Original undifferentiated MSCs derived from control and MPN mice grew continuously in undifferentiated culture conditions with a similar life expectancy, and both populations were capable of further differentiation into osteoblasts and chondrocytes. Collagen I (p<0.0001) and III (p=0.0008) mRNA and fiber formation were increased in the MSCs from mplW515L -transplanted mice compared to control. Without exogenous cytokines or HSC supernatant, the MSCs from mutant-mpl-mice maintained greater than 2-fold increase in collagen suggesting that intrinsic reprogramming occurred in the MSC population independent of tumor cells that are normally considered the source of TGFβ and other fibrosis-initiating cytokines. TGFβ mRNA (0.9878±0.129, control vs 1.023±0.319, MPN p=ns) and total TGFβ secreted protein detected by ELISA (165.0±22.7pg/ml, control vs 144.1±21.8pg/ml, MPN p=ns) was similar in control and MPN-derived MSCs indicating that excessive TGFβ production does not maintain the fibrotic phenotype in culture. We have shown previously that collagen from fibrotic MSCs in LGL leukemia is inhibited by culturing the cells with fibroblast growth factor-b (FGFb, FGF-2). Similar to LGL leukemia-dervied MSCs, the addition of FGFb stimulated proliferation and restored normal levels of collagen I and III in the MSCs from MPN mice.Conclusions: This study indicates that expanded MPN-derived MSCs can be re-programmed to produce normal amounts of collagen, which may reverse fibrosis. This study has implications for the development of MSC-based cell therapies in MPN. DisclosuresNo relevant conflicts of interest to declare.

Useful properties of undifferentiated mesenchymal stromal cells and adipose tissue as the source in liver-regenerative therapy studied in an animal model of severe acute fulminant hepatitis

Background aimsEnd-stage liver diseases frequently require liver transplantation. Cell therapy could be an alternative. This study aimed to analyze whether undifferentiated mesenchymal stromal cells (U-MSCs) or MSC-derived hepatocyte-like cells (DHLCs) from adipose tissue (AT), umbilical cord blood (UCB) and bone marrow (BM) would better restore damaged liver. MethodsAT was obtained from lipo-aspiration, UCB from an Umbilical Cord Blood Bank and BM from a BM Transplantation Unit. AT (collagenase digestion), UCB and BM (Ficoll gradient) were cultured (Dulbecco's modified Eagle's medium, low glucose, FBS) for 3 days. Detached adherent cells, at passage 4, were characterized as MSCs. Genetic stability was investigated by means of telomerase enzyme activity and karyotype. Hepatocyte differentiation protocol was performed with the use of Dulbecco's modified Eagle's medium, hepatocyte growth factor, basic fibroblast growth factor and nicotinamide (7 days); maturation medium (oncostatin, dexamethasone, insulin, transferrin and selenium) was added at 36 days. Hepatogenesis analyses were performed by use of morphology and albumin, AF, tyrosine-aminotransferase and glutamine synthetase gene expression and quantitative reverse transcription–polymerase chain reaction on days 9, 18, 25 and 36. Functionality was assessed through glycogen storage detection, indocyanine green absorption and transplantation procedure. U-MSCs and DHLCs were injected 48 h after induced fulminant hepatitis (intraperitoneal injection of carbon tetrachloride) in SCID/BALB-c mice. Histopathologic analyses were performed on days 7 and 15. Human origin included albumin and CK19 human markers. ResultsAll MSCs differentiated into functional hepatocyte-like cells, stored glycogen and absorbed indocyanine green. AT-MSC DHLC gene expression was more consistent with a normal hepatogenic-differentiation profile. UCB-MSCs expanded weakly, impairing their use for the transplantation procedure. AT and BM U-MSCs and DHLCs regenerated liver injury equally. Regenerated hepatocytes exhibited human origin. ConclusionsAT might be the source and U-MSCS the stem cells useful for liver-regenerative therapy.

Detection of hepatic maturation by raman spectroscopy in mesenchymal stem cells undergoing hepatic differentiation

Mesenchymal stromal cells (MSCs) are well known to have potential for applications in tissue engineering. In vitro, MSCs are able to differentiate to hepatocyte-like cells. The conventional methods to estimate the maturation of hepatic differentiation need large amount cells, cell fixation and labeling. In this study, the label-free method, Raman spectroscopy has used to real-time monitor the maturation level of hepatic differentiation from MSCs. The results indicated that the intensity of broad band in the range of 2800 cm-1 ∼ 3000 cm-1 reflecting glycogen within lipchromes in the MSCs-derived hepatocyte-like cells exhibited high correlation coefficient during maturation of hepatic differentiation. Furthermore, the principal component analysis of raman spectra (400 cm-1 ∼ 3000 cm-1) indicated the MSCs-derived hepatocyte-like cells were very different from the undifferentiated MSCs and close to the primary murine hepatocytes. In summary, Raman spectroscopy can provide a rapid, non-invasive, real-time and label-free biosensor and reflects the changes of live cell components during hepatic differentiation. Expectedly, these results can accelerate the improvements for clinical diagnosis and applications of stem cell research for tissue engineering.

MP19-13 DEVELOPMENT OF A LARGE XENOGRAFT ANIMAL MODEL TO INVSTIGATE EFFICACY OF CELL-BASED THERAPIES TO TREAT STRESS URINARY INCONTINENCE

You have accessJournal of UrologyStem Cell Research1 Apr 2015MP19-13 DEVELOPMENT OF A LARGE XENOGRAFT ANIMAL MODEL TO INVSTIGATE EFFICACY OF CELL-BASED THERAPIES TO TREAT STRESS URINARY INCONTINENCE Bastian Amend, Alexandra Kelp, Martin Vaegler, Arnulf Stenzl, and Karl-Dietrich Sievert Bastian AmendBastian Amend More articles by this author , Alexandra KelpAlexandra Kelp More articles by this author , Martin VaeglerMartin Vaegler More articles by this author , Arnulf StenzlArnulf Stenzl More articles by this author , and Karl-Dietrich SievertKarl-Dietrich Sievert More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.1005AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Currently, a functional cure for stress urinary incontinence (SUI) does not exist. The only feasible treatment is to ameliorate symptoms. Application of regeneration-competent progenitor or stem cells could possibly restore function of the rhabdosphincter. This study aims to develop a large animal model that incorporates safe, yet precise cell injection technologies, to investigate localization, distribution, survival, and possible integration of human mesenchymal stromal cells (MSC) after injection in the bladder sphincter muscle of minipigs. METHODS Undifferentiated different human MSC (bone marrow, placenta or adipose tissue) were injected into the rhabdosphincter (4 depots,2x106 cells in total) by transurethral-guided needle injection. Seventy-two immune-supressed female minipigs were divided into 12 groups with six animals each: 3 groups of MSC populations consisting of 4 follow-up points (3w,3m,6m,and 12m). Sham (24) underwent treatment with culture media. Functional regeneration of rhabdosphincter was evaluated by urodynamic urethral pressure profile(UPP) at above mentioned follow-up. The rhabdosphincter was harvested for further investigation. General histology was performed at the localization, distribution of MSC on cryostat sections as well as TUNEL-staining for the apoptosis rate. RESULTS Prior to initial MSC treatment, all animals received an MSC treatment Cyclosporine®(15mg/kg, iv) and continued orally post-operatively. Six did not survive; 5 from sham group and 1 that received cells. All animals tested (n=59/72) did not demonstrate signs of human MSC rejection nor inflammation. Only in 1/59, a fibrotic encapsulation of MSC injected was observed. The 59 urodynamic-investigated pigs demonstrated a functional increased UPP compared to sham without differences related to MSC source. During all tested evaluation points, cells remained in the region of injection without migration and/or disappearance. Over time the depots appeared to decrease. Throughout the investigation, apoptosis was present in ≤10% of injected MCS locations according to TUNEL-staining in the depots, whereas the host tissue had an average apoptosis of 2%. CONCLUSIONS Immune-supressed minipigs served as a model in preclinical cell therapy studies. Transurethral needle injection of human MSC is feasible and potential minimal-invasive application technique for clinical regeneration of injured and degenerated rhabdosphincter in the future, given that injected cells survived and effected functional regeneration. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e220 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Bastian Amend More articles by this author Alexandra Kelp More articles by this author Martin Vaegler More articles by this author Arnulf Stenzl More articles by this author Karl-Dietrich Sievert More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Chd1 Regulates Primitive State of Bone Marrow Mesenchymal Stromal Cells without Affecting Hematopoietic Support; An Insight on the Dissociation Between Primitive State and Niche Function

Mesenchymal stromal cells (MSCs) are characterized by heterogeneity in the proliferation/self-renewal potentials and hematopoietic supporting activity among subpopulations. Numerous studies have suggested that a primitive state of MSC subpopulation are correlated to its niche function to support hematopoietic stem cells (HSCs), but the mechanisms regulating primitive state of MSCs remains poorly understood.In the present study, we examined the role of a chromatin remodeling enzyme, chd1 in the maintenance of open chromatin and undifferentiated state of MSCs. We analyzed for expression in MSCs, the expression level of chd1 progressively decreased during in-vitro subculture (from 7 to 18 passages) in a manner proportional to the passage numbers. Moreover, chd1 expression was down regulated in the MSCs during their differentiation into adipogenic or osteogenic lineages, compared to proliferative state, indicating the correlations between MSC proliferation potentials and expression level of chd1. Next, we transduced human bone marrow-derived MSCs with shRNAs against chd1 and found that chd1 knock down MSCs (chd1-KD) exhibit significant loss of colony forming activity (CFU-F), decrease of cell proliferation and loss of multi-lineage differentiation towards osteogenic or adipogenic lineages. Moreover, chd1-KD MSCs exhibited lower level expression of pluripotency-related genes, oct-4, sox-2 and nanog, with concomitant increase of H3K9me3 on the promoters and decreased chromatin accessibility in the oct-4 promoter, suggesting that chd1 regulate open chromatin and multi-lineage potential of MSCs.However, KD of chd1 in MSCs did not affect the HSC-supporting activity of MSCs; human cord blood-derived CD34+ cells co-cultured on chd1-KD MSCs exhibited rather higher maintenance of primitive phenotype (CD34+90+) and higher repopulating activity in NOD/SCID-ɤC KO mice compared to those co-cultured on control group MSCs. Together, these results show that, while primitive state of MSCs are regulated by chromatin remodeling complex,chd1, the hematopoietic niche activity of MSCs is not directly influenced by the primitive state of MSCs, raising a questions on the prevailing notion that undifferentiated MSCs can better support hematopoietic function. DisclosuresNo relevant conflicts of interest to declare.

Efficacy of tissue engineered bone grafts containing mesenchymal stromal cells for cleft alveolar osteoplasty in a rat model

The development of sufficient tissue engineered bone grafts for alveolar cleft osteoplasty could reduce the necessity of autogenous bone grafts and its donor site morbidity. The aim of the study was to evaluate tissue engineered bone grafts in an artificially created bone defect.Bone grafts were created in vitro colonizing a synthetic hydroxyapatite–tricalciumphosphate scaffold (BONITmatrix®) with either undifferentiated mesenchymal stromal cells (group 1) or osteogenic differentiated mesenchymal stromal cells (group 2). Cells were multiplied from bone marrow of donor rats. Unmodified scaffolds (group 3) and the tissue engineered bone grafts were inserted into artificial maxillary defects of 54 Lewis rats. In 18 animals the defects remained unfilled (control). After one, three and six weeks the rats were sacrificed. The defect was evaluated radiologically and histologically with regard to the remaining defect volume and diameter. Statistical analysis followed.The bone grafts led to a specific bone formation at the defect margin. No complete reunion of any defect was observed within the healing time. After six weeks, the remaining defect volume was 6.86 ± 3.21 mm3 (control), 4.08 ± 1.36 mm3 (group 1), 5.00 ± 0.84 mm3 (group 2) 5.50 ± 1.05 mm3 (group 3). The remaining defect diameter measured 2.63 ± 0.52 mm (control), 2.39 ± 0.23 mm (group 1), 2.53 ± 0.22 mm (group 2) and 2.70 ± 0.66 mm (group 3). In all experimental groups the defect volume and diameter decreased over time, which was significant for group 1 (p = 0.014), group 2 (p = 0.025) and group 3 (p = 0.048). The defect volume and width was significantly reduced for bone grafts containing undifferentiated cells compared to control (p = 0.035) or scaffolds only (p = 0.05). ConclusionTissue engineered bone grafts induce a pronounced bone formation in artificial bone defects compared to unfilled controls or scaffolds only.

Chondrogenically differentiated mesenchymal stromal cell pellets stimulate endochondral bone regeneration in critical-sized bone defects

Grafting bone defects or atrophic non-unions with mesenchymal stromal cells (MSCs)-based grafts is not yet successful. MSC-based grafts typically use undifferentiated or osteogenically differentiated MSCs and regenerate bone through intramembranous ossification. Endochondral ossification might be more potent but requires chondrogenic differentiation of MSCs. Here, we determined if chondrogenically differentiated MSC (ch-MSC) pellets could induce bone regeneration in an orthotopic environment through endochondral ossification. Undifferentiated MSC pellets (ud-MSC) and ch-MSC pellets were generated from MSCs of human donors cultured on chondrogenic medium for respectively 3 (ud-MSC) and 21 (ch-MSC) days. A 6mm femoral bone defect was made and stabilised with an internal plate in 27 athymic rats. Defects were left empty for 6 weeks to develop an atrophic non-union before they were grafted with ch-MSC pellets or ud-MSC pellets. Micro-CT scans made 4 and 8 weeks after grafting showed that ch-MSC pellets resulted in significantly more bone than ud-MSC pellets. This regenerated bone could completely bridge the defect, but the amount of bone regeneration was donor-dependent. Histology after 7 and 14 days showed slowly mineralising pellets containing hypertrophic chondrocytes, as well as TRAP-positive and CD34-positive cells around the ch-MSC pellets, indicating osteoclastic resorption and vascularisation typical for endochondral ossification. In conclusion, grafting critical femoral bone defects with chondrogenically differentiated MSC pellets led to rapid and pronounced bone regeneration through endochondral ossification and may therefore be a more successful MSC-based graft to repair large bone defects or atrophic non-unions. But, since bone regeneration was donor-depend, the generation of potent chondrogenically differentiated MSC pellets for each single donor needs to be established first.