Human platelet lysate allows expansion and clinical grade production of mesenchymal stromal cells from small samples of bone marrow aspirates or marrow filter washouts

We compared two protocols for the expansion of human mesenchymal stromal cells (hMSCs) starting from diagnostic samples of BM aspirates (2-5 ml) or using the remnants in the bag and filter at the end of the BM infusions. The protocols differed in the presence of either 10% fetal bovine serum (FBS) or 5% platelet lysate (PL). We obtained a significantly (P=0.02) better expansion with PL, obtaining a median 1010-fold compared to 198-fold with a selected batch of FBS and in fewer days (29.8 in PL versus 41.4 in FBS). Overall, we recovered a variable number from 54.8 x 10(6) to 365 x 10(6) hMSCs in PL versus a variable number from 2.7 x 10(6) to 31 x 10(6) in FBS. No difference could be found in terms of gross morphology, differentiation potential, surface markers and immunological properties (inhibition of allogeneic PHA response and mixed lymphocyte reaction) of cells expanded with PL or FBS. The preparations were found within the range of acceptability for all the quality control criteria. Due to the clinical grade nature of the PL and the reproducibility of separate preparations, we propose this method to obtain hMSCs even from minute amounts of BM cells.     

Treatment of enterocutaneous fistula in Crohn’s Disease with adipose-derived stem cells: a comparison of protocols with and without cell expansion

Background  Expanded adipose-derived stem cells (ASC) have been shown to be effective in treating Crohn’s patients with enterocutaneous fistulas. It is possible that unexpanded cells corresponding to the stromal vascular fraction (SVF) may also be effective. Materials and methods  A subpopulation of patients from a previous proof-of-concept phase I study with enterocutaneous fistulas received autologous expanded ASCs. The same selection criteria for inclusion were applied to patients who underwent SVF implantation to treat enterocutaneous fistulas. After tract curettage, cell suspensions (either SVF cells from lipoaspirate or expanded ASCs) were injected into the tract walls, and the fistulous tract was sealed with fibrin adhesive (with or without cells). Results  In the series that received ASCs, four fistulas could be evaluated, and cure was achieved in three out of four cases. In the series that received SVF cells, four fistulas were evaluated, with cure achieved in one out of four cases. Conclusions  Although a comparison of case series cannot be considered firm evidence, a therapeutic protocol that uses expansion prior to implantation does seem to be more effective than one that uses SVF cells directly from a lipoaspirate sample.     

Efficient replacing of fetal bovine serum with human platelet releasate during propagation and differentiation of human bone-marrow-derived mesenchymal stem cells to functional hepatocyte-like cells

Objectives  The aim of this study was to find out substitution effect of fetal bovine serum (FBS) with human platelet releasate (HPR) as a major growth factor source during expansion and differentiation of human bone marrow-derived mesenchymal stem cells (hBMSC) into hepatocytes.
Methods  Propagation and differentiation potential of hBMSCs into hepatocyte-like cells in a medium fortified with HPR instead of FBS were investigated with morphological, cytochemical and molecular experiments.
Results  Multiplex analysis showed that HPR was more efficient than FBS in supporting hBMSC outgrowth. The proliferation rate of MSC in presence of HPR (derived from 10⁹ platelets/ml) was about threefold greater than that of FBS ( P <  0·001). Despite the differences in MTT value, hBMSCs-driven HPR or FBS did not differ in terms of gross morphology, immunophenotype and osteogenic differentiation potential. Hepatic differentiation of hBMSCs was successfully performed in the media enriched with HPR. Immunoreactivity of cells with monoclonal antibodies against for albumin and α-fetoprotein (AFP) was even more positive in hepatocytes differentiated in presence of HPR as compared to that of FBS. The gene expression of albumin, AFP and cytokeratin-18 at mRNA levels in differentiated cells attest to supporting role of HPR in hepatic differentiation media. These findings were further confirmed with greater urea production (approximately twofold) in the culture media of cells differentiated under HPR compared to that in FBS ( P <  0·001).
Conclusion  Human platelet releasate is an efficient and safe substitute for FBS in culture media used for expansion and differentiation of hBMSCs to hepatocyte.     

Intracoronary and retrograde coronary venous myocardial delivery of adipose‐derived stem cells in swine infarction lead to transient myocardial trapping with predominant pulmonary redistribution

Objectives: To examine the comparative fate of adipose‐derived stem cells (ASCs) as well as their impact on coronary microcirculation following either retrograde coronary venous (RCV) or arterial delivery. Background: Local delivery of ASCs to the heart has been proposed as a practical approach to limiting the extent of myocardial infarction. Mouse models of mesenchymal stem cell effects on the heart have also demonstrated significant benefits from systemic (intravenous) delivery, prompting a question about the advantage of local delivery. There has been no study addressing the extent of myocardial vs. systemic disposition of ASCs in large animal models following local delivery to the myocardium. Methods: In an initial experiment, dose‐dependent effects of ASC delivery on coronary circulation in normal swine were evaluated to establish a tolerable ASC dosing range for intracoronary (IC) delivery. In a set of subsequent experiments, an anterior acute myocardial infarction (AMI) was created by balloon occlusion of the proximal left anterior descending (LAD) artery, followed by either IC or RCV infusion of 10^{7 111}Indium‐labeled autologous ASCs 6 days following AMI. Indices of microcirculatory resistance (IMR) and coronary flow reserve (CFR) were measured before sacrifices to collect tissues for analysis at 1 or 24 hr after cell delivery. Results: IC delivery of porcine ASCs to normal myocardium was well tolerated up to a cumulative dose of 14 × 10⁶ cells (approximately 0.5 × 10⁶ cells/kg). There was evidence suggesting microcirculatory trapping of ASC: at unit doses of 50 × 10⁶ ASCs, IMR and CFR were found to be persistently altered in the target LAD distribution at 7 days following delivery, whereas at 10 × 10⁶ ASCs, only CFR was altered. In the context of recent MI, a significantly higher percentage of ASCs was retained at 1 hr with IC delivery compared with RCV delivery (57.2 ± 12.7% vs. 17.9 ± 1.6%, P = 0.037) but this initial difference was not apparent at 24 hr (22.6 ± 5.5% vs. 18.7 ± 8.6%; P = 0.722). In both approaches, most ASC redistributed to the pulmonary circulation by 24 hr postdelivery. There were no significant differences in CFR or IMR following ASC delivery to infarcted tissue by either route. Conclusions: Selective intravascular delivery of ASC by coronary arterial and venous routes leads to similarly limited myocardial cell retention with predominant redistribution of cells to the lungs. IC arterial delivery of ASC leads to only transiently greater myocardial retention, which is accompanied by obstruction of normal regions of coronary microcirculation at higher doses. The predominant intrapulmonary localization of cells following local delivery via both methods prompts the notion that systemic delivery of ASC might provide similarly beneficial outcomes while avoiding risks of inadvertent microcirculatory compromise. © 2012 Wiley Periodicals, Inc.     

Efficacy of adipose tissue-derived mesenchymal stem cells for fulminant hepatitis in mice induced by concanavalin A

Background and Aim: Fulminant hepatitis is mainly caused by excessive immune response‐mediated liver injury and its definitive therapy is liver transplantation. Mesenchymal stem cells, one of the adult stem cells, have an immunomodulatory effect on immune cells and reside in various tissues. The aim of this study was to investigate a therapeutic effect of adipose tissue‐derived mesenchymal stem cells (ASCs) on fulminant hepatitis induced by concanavalin A (ConA). Methods: The ASCs were isolated from adipose tissues of BALB/c mice and confirmed by detection of cell surface markers and induction of multi‐lineage differentiation. BALB/c mice were injected with ConA and treated with ASCs, phosphate buffered saline (PBS) or splenocytes (SPLCs). Survival rates, levels of serum liver enzymes, titers of serum cytokines, histopathology and localization of ASCs were investigated. Result: The survival rate of ASC‐injected mice significantly increased compared to PBS or SPLC‐injected mice. This effect was dependent on doses and timing of ASCs injected. Improvement of liver enzyme levels, histopathological changes and suppression of inflammatory cytokine production were observed in ASC‐injected mice. Fluorescent stained ASCs were detected in inflammatory liver, but not in normal liver. Conclusion: These results suggest that ASC treatment has a high potential to be an innovative therapy for fulminant hepatitis.     

Cellular mechanical properties reflect the differentiation potential of adipose-derived mesenchymal stem cells

The mechanical properties of adipose-derived stem cell (ASC) clones correlate with their ability to produce tissue-specific metabolites, a finding that has dramatic implications for cell-based regenerative therapies. Autologous ASCs are an attractive cell source due to their immunogenicity and multipotent characteristics. However, for practical applications ASCs must first be purified from other cell types, a critical step which has proven difficult using surface-marker approaches. Alternative enrichment strategies identifying broad categories of tissue-specific cells are necessary for translational applications. One possibility developed in our lab uses single-cell mechanical properties as predictive biomarkers of ASC clonal differentiation capability. Elastic and viscoelastic properties of undifferentiated ASCs were tested via atomic force microscopy and correlated with lineage-specific metabolite production. Cell sorting simulations based on these "mechanical biomarkers" indicated they were predictive of differentiation capability and could be used to enrich for tissue-specific cells, which if implemented could dramatically improve the quality of regenerated tissues.     

Platelet lysate as a serum replacement for skin cell culture on biomimetic PCL nanofibers

Platelets are a popular source of native growth factors for tissue engineering applications. The aim of the study was to verify the use of platelet lysate as a fetal bovine serum (FBS) replacement for skin cell culture. The cytokine content of the platelet lysate was characterized using the Bio-Plex system. The cells (fibroblasts, melanocytes, and keratinocytes) were cultured on PCL nanofibrous scaffolds to mimic their natural microenvironment. The cytokine content of the platelet lysate was determined, and to the cells, a medium containing platelet lysate or platelet lysate in combination with FBS was added. The results showed that 7% (v/v) platelet lysate was sufficient to supplement 10% (v/v) FBS in the culture of fibroblasts and keratinocytes. The combination of platelet lysate and FBS had a rather inhibitory effect on fibroblasts, in contrary to keratinocytes, where the effect was synergic. Platelet lysate did not sufficiently promote proliferation in melanocytes; however, the combination of FBS and platelet lysate yielded a better outcome and resulted in bipolar morphology of the cultured melanocytes. The data indicated that platelet lysate improved cell proliferation and metabolic activity and may be used as an additive to the cell culture media.     

The modified International Prognostic Index can predict the outcome of localized primary intestinal lymphoma of both extranodal marginal zone B-cell and diffuse large B-cell histologies

This study aimed to assess the potential of human cord blood (CB) cells to engraft in the xenogenic non-obese diabetic/severe combined immunodeficient (NOD/SCID) mouse model after in vitro expansion culture. We also studied the quality of human haemopoiesis arising from the transplantation of fresh or expanded cells in this model. Cord blood CD34(+) cells were cultured for 3, 7 or 10 d with stem cell factor, Flt3, thrombopoietin, interleukin 3 (IL-3), IL-6 and granulocyte colony-stimulating factor, all at 10 ng/ml in serum-replete conditions. Transplantation of mice with fresh CB containing 3 x 10(4) CD34(+) cells and 1-2 SCID repopulating cells (SRC) resulted in a median of 7.4% (0.4%-76.8%) human engraftment. When mice received the expanded product of 1-2 SRC, the ability to repopulate NOD/SCID mice was maintained even after 10 d of in vitro culture. Serial dilution of the expanded cells suggested that in vitro expansion had increased SRC numbers two- to fourfold. Expanded SRC produced long-term culture-initiating cells, clonogenic cells and CD34(+) cells in the same proportions as fresh cells after successful engraftment. Therefore, expanded SRC were able to differentiate in the same way as fresh SRC. There was a trend towards lower levels of engraftment when d 7 cultured cells were transplanted (median engraftment 0.8%, range 0.0-24.0%) compared with 1-2 fresh SRC. Our data suggest that this is owing to reduced proliferation of cultured cells in vivo. By utilizing limiting numbers of CB SRC, we confirmed that the engraftment potential of SRC in the NOD/SCID model was preserved after in vitro expansion. Furthermore, dilution experiments strongly suggest two- to fourfold expansion of SRC in vitro. These studies are relevant for developing clinical stem cell expansion strategies.     

Reduced social interaction and ultrasonic communication in a mouse model of monogenic heritable autism

Autism spectrum conditions (ASCs) are heritable conditions characterized by impaired reciprocal social interactions, deficits in language acquisition, and repetitive and restricted behaviors and interests. In addition to more complex genetic susceptibilities, even mutation of a single gene can lead to ASC. Several such monogenic heritable ASC forms are caused by loss-of-function mutations in genes encoding regulators of synapse function in neurons, including NLGN4. We report that mice with a loss-of-function mutation in the murine NLGN4 ortholog Nlgn4, which encodes the synaptic cell adhesion protein Neuroligin-4, exhibit highly selective deficits in reciprocal social interactions and communication that are reminiscent of ASCs in humans. Our findings indicate that a protein network that regulates the maturation and function of synapses in the brain is at the core of a major ASC susceptibility pathway, and establish Neuroligin-4-deficient mice as genetic models for the exploration of the complex neurobiological disorders in ASCs.     

Circulating mature NK (Leu11+) cells positive for IL2 receptor and HLA-DR in patients with various liver diseases and asymptomatic HBsAg carriers

IL2R+ Leu11+ cells (A) and HLADR+ Leu11+ cells (B) of peripheral blood in patients with various liver diseases and ASCs were measured using double colour immunofluorescence assay of MoAb with FACS flow cytometry. 1) The mean % of IL2R+ Leu11+ cells which was 0.5 +/- 0.2 in healthy controls, decreased significantly in HCC in comparison with CALD, ASC and healthy controls, and in ASC rather than in healthy controls. They (A) were less than 0.1% in eight of thirteen cases with HCC, in one of twenty cases with CALD and of eleven ASCs, respectively. 2) In the mean % of HLADR+ Leu11+ cells which was 2.9 +/- 1.8 in healthy controls there was not a significant difference among HCC, CALD, ASC, AH and healthy controls. They (B) were less than 0.1% only in one case with HCC. 3) The value of fluorescence intensity of IL2R on IL2R+ Leu11+ cells reduced in B-CALD, ASC, AH and HCC, and one of HLADR on HLADR+ Leu11+ cells increased in CALD. These results suggested that the decrease of IL2R+ Leu11+ cells was due to the existence of HCC.     

Ex vivo T lymphocyte expansion for retroviral transduction: influence of serum-free media on variations in cell expansion rates and lymphocyte subset distribution

OBJECTIVE: In the setting of allogeneic stem cell transplantation, suicide gene-manipulated donor T cells that can be selectively inactivated in vivo would potentially allow optimal control of the GVL (graft-vs-leukemia)/GVHD (graft-vs-host disease) balance. Retroviral T-cell transduction requires ex vivo cell expansion, which is often achieved by IL-2 and anti-CD3 stimulation. Traditionally, culture media for cell expansion are supplemented with fetal bovine serum (FBS) or human serum. While these sera promote cell growth and viability, they contain uncharacterized elements that may yield inconsistent results from batch to batch. Cell expansion in serum-free media would therefore be preferable. MATERIALS AND METHODS: We compared T-cell expansion rates in three commercially available serum-free culture media (X-VIVO 15, AIM-V, and Cellgro SCGM), with or without the addition of human serum (HS, 5%). We also aimed to evaluate how the in vitro expansion affected the composition of the various T-cell subsets. Buffy-coats from four healthy donors were expanded for 21 days. The media were compared to standard RPMI 1640 medium, supplemented with HS (5%) or FBS (10%). For retroviral transductions, the LN vector carrying the neomycin- resistance gene was used in four additional donors. RESULTS: In our hands, X-VIVO 15 gave the highest rate of serum-free expansion (a median of 79-fold expansion, range 20-117). For serum-free expansion, activation with OKT3 for 21 days gave slightly higher expansion rates than a 5-day course (however, without statistical significance). When serum was added, this discrepancy was not seen. Cytokine analysis (IFN-gamma, IL-10, and IL-4) showed a distinct type1 cytokine pattern with elevated IFN-gamma levels during the whole period of culture. Flow cytometric analyses showed substantial inter-media, but also some inter-donor, variability in T-cell subset compositions. Transduction of cells with the LN vector and G418 selection resulted in a 14-fold increase (range 3-18) for serum-free X-VIVO 15 based cultures. Cell phenotypes remained unchanged by the transduction procedure as compared to nontransduced cells. CONCLUSION: Among the tested serum-free media, X-VIVO 15 has shown to best support the in vitro expansion of T cells, resulting in equal percentages of CD4(+) and CD8(+) cells. These cells can easily be transduced and selected. There seem to be no significant benefits, regarding absolute cell numbers or T-cell subset compositions, with OKT3-stimulation for more than five days. The addition of low levels of HS increases the consistencies in the cell expansion rates for all media.     

Implanted adipose progenitor cells as physicochemical regulators of breast cancer

Multipotent adipose-derived stem cells (ASCs) are increasingly used for regenerative purposes such as soft tissue reconstruction following mastectomy; however, the ability of tumors to commandeer ASC functions to advance tumor progression is not well understood. Through the integration of physical sciences and oncology approaches we investigated the capability of tumor-derived chemical and mechanical cues to enhance ASC-mediated contributions to tumor stroma formation. Our results indicate that soluble factors from breast cancer cells inhibit adipogenic differentiation while increasing proliferation, proangiogenic factor secretion, and myofibroblastic differentiation of ASCs. This altered ASC phenotype led to varied extracellular matrix (ECM) deposition and contraction thereby enhancing tissue stiffness, a characteristic feature of breast tumors. Increased stiffness, in turn, facilitated changes in ASC behavior similar to those observed with tumor-derived chemical cues. Orthotopic mouse studies further confirmed the pathological relevance of ASCs in tumor progression and stiffness in vivo. In summary, altered ASC behavior can promote tumorigenesis and, thus, their implementation for regenerative therapy should be carefully considered in patients previously treated for cancer.     

Adipose-derived stem cells on the healing of ischemic colitis: a therapeutic effect by angiogenesis

Background

There has been no specific treatment for ischemic colitis. We verified the effects of adipose-derived stem cells (ASCs) on ischemia-induced colitis in a rat model.

Methods

Forty male Sprague–Dawley rats (10?weeks old; weight, 350?±?20?g) were divided into two groups: a control group (only fibrinogen and thrombin injected, n?=?20) and an ASC group (local implantation of ASCs mixed with thrombin and fibrinogen, n?=?20). An ischemic colitis model was established by modifying Nagahata's methods with double-blind randomization. ASCs (1?×?10⁶ cells) were implanted intramurally into the ischemic area using a fibrin glue mixture. The severity of adhesion, degree of ileus, the number and size of the ulcers, Wallace macroscopic and microscopic scores, and microvascular density were measured.

Results

The degree of ileus was significantly lower, and significantly fewer and smaller ulcerations were found in the ASC group than those in the control group. Wallace macroscopic and microscopic scores were lower in the ASC group than in the control group (1.90?±?1.22 versus 3.25?±?1.83, p?<?0.01 and 1.55?±?1.88 versus 2.84?±?1.89, p?<?0.05, respectively). Microvascular density was higher in the ASC group than in the control (54.45?±?19.45 versus 26.54?±?13.14, p?<?0.01, respectively).

Conclusions

Local implantation of ASCs into an ischemic-injured colonic wall reduced the grade of ischemic injury and enhanced tissue healing by promoting angiogenesis.     

3种制备方法对血小板活化状态的影响

目的：研究富血小板血浆法（PRP）、白膜回浆法（BC）和血细胞分离机（Trima）单采3种制备方法对血小板活化状态的影响。方法：采用流式细胞术和酶联免疫吸附法分别检测3种方法制备的血小板表面CD62P和血浆可溶性CD62P（sCD62P）的表达。结果：Trima制备的单采血小板CD62P和血浆sCD62P的表达水平均明显低于BC和PRP制备的浓缩血小板（P〈0.01）。在3种血小板制品中,PRP制备的浓缩血小板CD62P和sCD62P的表达水平最高。结论：血细胞分离机（Trima）制备的单采血小板活化水平明显低于BC和PRP制备的浓缩血小板。     

Deficient primary cilia in obese adipose‐derived mesenchymal stem cells: obesity,a secondary ciliopathy?

Obesity alters the composition, structure and function of adipose tissue, characterized by chronic inflammation, insulin resistance and metabolic dysfunction. Adipose‐derived mesenchymal stem cells (ASCs) are responsible for cell renewal, spontaneous repair and immunomodulation in adipose tissue. Increasing evidence highlights that ASCs are deficient in obesity, and the underlying mechanisms are not well understood. We have recently shown that obese ASCs have defective primary cilia, which are shortened and unable to properly respond to stimuli. Impaired cilia compromise ASC functions. This work suggests an intertwined connection of obesity, defective cilia and dysfunctional ASCs. We have here discussed the current data regarding defective cilia in various cell types in obesity. Based on these observations, we hypothesize that obesity, a systemic chronic metainflammation, could impair cilia in diverse ciliated cells, like pancreatic islet cells, stem cells and hypothalamic neurons, making these critical cells dysfunctional by shutting down their signal sensors and transducers. In this context, obesity may represent a secondary form of ciliopathy induced by obesity‐related inflammation and metabolic dysfunction. Reactivation of ciliated cells might be an alternative strategy to combat obesity and its associated diseases.     

Classifying acute leukemia by immunophenotyping: a combined FAB-immunologic classification of AML

A panel of commercially available monoclonal antibodies and five heteroantisera were used to distinguish and subtype 138 cases of acute leukemia (AL). The immunophenotype was compared with the French-American-British (FAB) classification obtained on the cases. The immunophenotype discriminated acute myelogenous leukemia (AML) from acute lymphoblastic leukemia (ALL) and recognized cases not distinguished by cytochemistry (22% of cases), mixed lineage phenotypes (13% of cases), and cases with separate populations of lymphoblasts and myeloblasts (one case). Using the immunologic panel and derived criteria to subtype AML, correspondence of the immunophenotype to the FAB subtypes M1, M2, M4, and M5 was possible in greater than 80% of cases. A combined classification of the immunophenotype and FAB morphology/cytochemistry was devised for AML subtyping. It is recommended that immunophenotyping should be done at least in all cases with negative or inconclusive cytochemistry. At present, we suggest that until a "gold standard" for identifying leukemic subtypes is developed, the best method for typing acute leukemia is by using a combination of morphology, cytochemistry and immunophenotyping.     

Establishment,growth properties,and morphological characteristics of permanent human small cell lung cancer cell lines

Summary Cell lines from SCLC were established with a success rate of 43% from different metastatic sites of treated and untreated patients. All 6 SCLC cell lines grew as floating cell aggregates without substrate adherence. The degree of aggregation ranged from very tight spheroids to very loose sheets and chains. This gross morphological property showed a striking correlation to the PDT, with short PDTs in loose growing cell lines and long PDTs in tight growing cell lines. Cell size and nuclear features, i.e., chromatin pattern and nucleolar prominence, also seemed to correlate with the PDT and gross morphology. All SCLC cell lines had dense core granules by electron microscopical examination. Several different serum-free and serum-supplemented growth media were tested for their feasibility in estabilishing and permanently growing SCLC. Serum-free SIT medium and SIT 2.5 medium provided the best results in liquid culture. For semisolid SCLC cultivation, R10 medium was suprior to all other media tested. These cell lines are currently under intensive biochemical, molecular biological, and cytogenetical investigation in different laboratories and thus provide a tool for studying the biology of lung cancer.Abbreviations SCLC small cell lung cancer - FBS fetal bovine serum - R 10 Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS - D 10 Dulbecco's modified Eagle's medium supplemented with 10% FBS - SIT RPMI 1640 medium supplemented with selenium, insulin, transferrin - HITES RPMI 1640 medium supplemented with hydrocortisone, insulin, transferrin, estradiol, selenium - SIT 2.5 SIT medium supplemented with 2.5% FBS - PBS phosphate buffered saline - PDT population doubling time This work was supported by the SFB 215 of the German Research Society. The authors Thank Dr. Adi F. Gazdar for reviewing the morphology     

Human marrow mesenchymal stem cell culture: serum-free medium allows better expansion than classical alpha-MEM medium

The expansion of mesenchymal stem cells (MSCs) strongly depends on the culture conditions and requires medium supplemented with 10-20% fetal calf serum (FCS) to generate relevant numbers of cells. However, the presence of FCS is a major obstacle for their clinical use. Therefore, we have evaluated the capacity of expansion of MSC in a commercial serum-free medium (UC) supplemented with a serum substitute (ULTROSER) in comparison with a classical medium alpha-MEM containing 15% FBS. Bone marrow-mononuclear cells collected from 12 volunteer healthy donors were expanded in two different culture media. MSCs isolated in the both media were morphologically similar and expressed identical phenotypic markers. After the primoculture (P0) and one passage, we obtained significantly more MSC and CFU-F progenitors in UC medium than in alphaMEM. Their multipotentiality was preserved during culture, as well as their capacity to support haematopoiesis. In conclusion, our observations strongly suggest that UC is an optimal medium for ex vivo expansion of MSC: it allows a better cell expansion, preserves cell multipotentiality, reduces the culture period and contains low concentration of serum substitute. This medium seems suitable for clinical scale expansion of MSC.     

Adipose-Derived Stem Cells Ameliorate Experimental Murine Colitis via TSP-1-Dependent Activation of Latent TGF-β

Background

Adipose tissue-derived stem cells (ASCs) have been investigated as therapeutic tools for a variety of autoimmune diseases, including inflammatory diseases. However, the mechanisms underlying the immunomodulatory properties of ASCs are not well understood. Here, we investigated the mechanism of regulatory T cell (Treg) induction in ASC therapy in a murine model of inflammatory bowel disease.

Methods

Acute colitis was induced in mice using dextran sulfate sodium and ASCs administered intraperitoneally. Tregs and CD103⁺ dendritic cells were analyzed in the mesenteric lymph nodes (MLNs), spleen, and colonic lamina propria (CLP). Activation of latent TGF-β by ASCs was analyzed in vitro using ELISA. siRNA technology was used to create ASCs in which TSP-1 or integrinαv was knocked down in order to investigate the involvement of these proteins in the activation of latent TGF-β. In addition, TSP-1-knockdown ASCs were administered to mice with colitis to assess their clinical efficacy in vivo.

Results

Systemic administration of ASCs significantly lessened the clinical and histopathological severity of colitis. ASCs were distributed throughout the lymphatic system in the MLNs and spleen. Tregs were increased in the MLNs and CLP, but CD103⁺ dendritic cells were not significantly altered. The ASCs activated latent TGF-β. TSP-1 knockdown impaired TGF-β activation in vitro and abrogated the therapeutic effects of the ASCs in vivo. Furthermore, Tregs were not increased in the MLNs and CLP from mice treated with TSP-1-knockdown ASCs.

Conclusions

These results demonstrate that ASCs induce Tregs by activating latent TGF-β via TSP-1, independent of CD103⁺ dendritic cell induction.