In vitro differentiation of bone marrow derived porcine mesenchymal stem cells to endothelial cells

Mesenchymal stem cells (MSCs) hold potential for the regeneration of damaged tissues in cardiovascular diseases. In this study, we investigated the potential of porcine MSCs to differentiate into endothelial cells (ECs) in vitro. The cultured bone marrow‐derived cells were CD11b^–CD34^–CD44⁺CD45^–CD90⁺ and showed mesodermal lineage differentiation, which is characteristic of MSCs. The MSCs were induced to differentiate into ECs using endothelial growth medium (EGM), with and without high concentrations of VEGF (EGM + VEGF; 50 ng/ml). Endothelial basal medium (EBM) without growth factors served as the control. The EC differentiation was assessed by the presence of vWF, ability to take up acetylated LDL, in vitro angiogenesis assay, flow cytometry and qPCR of EC markers vWF, VE‐cadherin, PECAM‐1, VEGF‐R1 and VEGF‐R2 after 10 days of stimulation. Cells cultured in EGM + VEGF medium demonstrated higher amounts of DiI‐AcLDL‐positive cells and enhanced the presence of vWF (90%), VE‐Cadherin‐ (60%) and PECAM‐1 (48%)‐positive cells, than in EBM. These cells showed profuse sprouting of capillary tubes and closed polygon formation in the angiogenesis assay. There was 1.5–2‐fold increase in the mRNA expression of endothelial markers in the cells stimulated with EGM + VEGF medium when compared to control. The results demonstrate the ability of porcine MSCs to differentiate into ECs under in vitro inducing conditions. The differentiated cells would provide new options for re‐endothelialization following interventional procedures and tissue engineering. Copyright © 2012 John Wiley & Sons, Ltd.     

Mesenchymal stem cells from CD34− human umbilical cord blood

Umbilical cord blood (UCB) is well known to be a rich source of stem cells especially for haematopoietic stem cells (HSCs). Recently, mesenchymal stem cells (MSCs) have also been shown to exist in cord blood. Although MSCs have been described by a subset of surface antigens after expansion, little is known about the cell surface phenotype of undifferentiated MSCs. The aim of this study therefore was to clarify whether undifferentiated MSCs are resident among CD34^? UCB cells. CD34⁺ cells were separated from UCB mononuclear cells (MNCs) by magnetic sorting and the CD34^? cell fractions were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% foetal calf serum (FCS) and basic‐fibroblast growth factor. Isolated CD34⁺ cells were also cultured in the same medium. Adherent fibroblast‐like cells at passage 3–4 were analyzed by fluorescence‐activated cell sorting (FACS) for MSC marker expression , and standard adipogenic, osteogenic and chondrogenic assays were used to investigate their differentiation potentials. After 4–5 weeks in culture, the cells from the CD34^? fraction became confluent with flat and fibroblast‐like morphology. These cells were positively stained for the mesenchymal cell markers CD29, CD73 and CD105. In adipogenic differentiation, the cells showed oil red O positive and expressed FABP4, adipsin and proliferation‐activated receptor γ‐2 (PPARγ2 genes) associated with adipogenesis. In osteogenic differentiation, calcium accumulation and osteocalcin were detected. The cells grown in chondrogenic conditions were positively stained for human aggrecan and expressed collagen type II and Sox‐9 genes. In contrast, cells from the CD34⁺ fraction failed to generate any cells with MSC morphology under the same culture conditions. Our results showed that UCB contained MSCs which are only resident in the CD34^? fraction. The MSCs could be induced to differentiate into at least three lineage cell types, adipocytes, osteoblasts and chondrocytes.     

Engineered extracellular matrix components do not alter the immunomodulatory properties of mesenchymal stromal cells in vitro

Mesenchymal stromal cells (MSCs) have emerged as promising candidates for regenerative therapies, including tissue engineering. Recently it has been reported that engineered extracellular matrix (ECM) components support the differentiation of MSCs into osteocytes and chondrocytes, indicating that ECM components may represent attractive carriers for MSC transplants to repair damaged tissues. However, little is known about the impact of engineered ECM components on the immunosuppressive properties of MSCs, which may essentially contribute to the prevention of allogeneic MSC transplant rejection. In the present study, we explored the potential of fibronectin, fibrillar collagen I, tropocollagen and collagen I/heparin to influence the immunosuppressive capacities of MSCs. We found that these ECM components do not modulate the capability of MSCs to inhibit the proliferation of anti‐CD3/anti‐CD28 antibody‐stimulated CD4⁺ and CD8⁺ T cells and of lymphocytes in a mixed lymphocyte reaction. In addition, the potential of MSCs to impair the production of immunostimulatory IL‐12 and to improve the release of immunosuppressive IL‐10 by 6‐sulpho LacNAc⁺ (slan) dendritic cells (DCs), representing a pro‐inflammatory subset of human blood DCs, was not altered by the ECM components. Furthermore, ECM components do not influence the ability of MSCs to inhibit the slanDC‐induced proliferation of CD4⁺ T cells. In conclusion, the used engineered ECMs maintain important immunosuppressive properties of MSCs, which support their suitablility as carriers for MSC transplants in tissue engineering. Copyright © 2012 John Wiley & Sons, Ltd.     

CD45/CD11b positive subsets of adult lung anchorage‐independent cells harness epithelial stem cells in culture

Compensatory growth is mediated by multiple cell types that interact during organ repair. To elucidate the relationship between stem/progenitor cells that proliferate or differentiate and somatic cells of the lung, we used a novel organotypic ex vivo pneumoexplant system. Applying this technique, we identified a sustained culture of repopulating adult progenitors in the form of free‐floating anchorage‐independent cells (AICs). AICs did not express integrin proteins α5, β3 and β7, and constituted 37% of the total culture at day 14, yielding a mixed yet conservative population that recapitulated RNA expression patterns of the healthy lung. AICs exhibited rapid proliferation manifested by a marked 60‐fold increase in cell numbers by day 21. More than 50% of the AIC population was c‐KIT⁺ or double‐positive for CD45⁺ and CD11b⁺ antigenic determinants, consistent with cells of hematopoietic origin. The latter subset was found to be enriched with prosurfactant protein‐C and SCGB1A1 expressing putative stem cells and with aquaporin‐5 producing cells, characteristic of terminally differentiated alveolar epithelial type‐1 pneumocytes. At the air/gel interface, AICs undergo remodeling to form a cellular lining, whereas TGF(β)1 treatment modifies protein expression properties to further imply a robust effect of the microenvironment on AIC phenotypic changes. These data confirm the active participation of clonogenic hematopoietic stem cells in a mammalian model of lung repair and validate mixed stem/somatic cell cultures, which license sustained cell viability, proliferation and differentiation, for use in studies of compensatory pulmonary growth. Copyright © 2012 John Wiley & Sons, Ltd.     

Dynamic cell–cell interactions between cord blood haematopoietic progenitors and the cellular niche are essential for the expansion of CD34+, CD34+CD38− and early lymphoid CD7+ cells

Most clinical applications of haematopoietic stem/progenitor cells (HSCs) would benefit from their ex vivo expansion to obtain a therapeutically significant amount of cells from the available donor samples. We studied the impact of cellular interactions between umbilical cord blood (UCB) haematopoietic cells and bone marrow (BM)‐derived mesenchymal stem cells (MSCs) on the ex vivo expansion and differentiative potential of UCB CD34⁺‐enriched cells. UCB cells were cultured: (a) directly in contact with BM MSC‐derived stromal layers (contact); (b) separated by a microporous membrane (non‐contact); or (c) without stroma (no stroma). Highly dynamic culture events occurred in HSC‐MSC co‐cultures, involving cell–cell interactions, which preceded HSC expansion. Throughout the time in culture [18 days], total cell expansion was significantly higher in contact (fold increase of 280 ± 37 at day 18) compared to non‐contact (85 ± 25). No significant cell expansion was observed in stroma‐free cultures. CD34⁺ cell expansion was also clearly favoured by direct contact with BM MSCs (35 ± 5‐ and 7 ± 3‐fold increases at day 18 for contact and non‐contact, respectively). Moreover, a higher percentage of CD34⁺CD38^? cells was consistently maintained during the time in culture under contact (8.1 ± 1.9% at day 18) compared to non‐contact (5.7 ± 1.6%). Importantly, direct cell interaction with BM MSCs significantly enhanced the expansion of early lymphoid CD7⁺ cells, yielding considerably higher (×3–10) progenitor numbers compared to non‐contact conditions. These results highlight the importance of dynamic cell–cell interactions between UCB HSCs and BM MSCs, towards the maximization of HSC expansion ex vivo to obtain clinically relevant cell numbers for multiple settings, such as BM transplantation or somatic cell gene therapy. Copyright © 2009 John Wiley & Sons, Ltd.     

Ex vivo expansion of cord blood haematopoietic stem/progenitor cells under physiological oxygen tensions: clear‐cut effects on cell proliferation,differentiation and metabolism

Physiologically low O₂ tensions are believed to regulate haematopoietic stem cell (HSC) functions in the bone marrow (BM; 0–5%). In turn, placenta and umbilical cord are characterized by slightly higher physiological O₂ tensions (3–10%). We hypothesized that O₂ concentrations within this range may be exploited to augment the ex vivo expansion/maintenance of HSCs from umbilical cord (placental) blood (UCB). The expansion of UCB CD34⁺‐enriched cells was studied in co‐culture with BM mesenchymal stem/stromal cells (MSCs) under 2%, 5%, 10% and 21% O₂. 2% O₂ resulted in a significantly lower CD34⁺ cell expansion (25‐fold vs 60‐, 64‐ and 92‐fold at day 10 for 5%, 21%, 10% O₂, respectively). In turn, 10% O₂ promoted the highest CD34⁺CD90⁺ cell expansion, reaching 22 ± 5.4‐ vs 5.6 ± 2.4‐ and 5.7 ± 2.0‐fold for 2%, 5% and 21% O₂, respectively, after 14 days. Similar differentiation patterns were observed under different O₂ tensions, being primarily shifted towards the neutrophil lineage. Cell division kinetics revealed a higher proliferative status of cells cultured under 10% and 21% vs 2% O₂. Expectedly, higher specific glucose consumption and lactate production rates were determined at 2% O₂ when compared to higher O₂ concentrations (5–21%). Overall, these results suggest that physiological oxygen tensions, in particular 10% O₂, can maximize the ex vivo expansion of UCB stem/progenitor cells in co‐culture with BM MSCs. Importantly, these studies highlight the importance of exploiting knowledge of the intricate microenvironment of the haematopoietic niche towards the definition of efficient and controlled ex vivo culture systems capable of generating large HSCs numbers for clinical applications. Copyright © 2013 John Wiley & Sons, Ltd.     

Skin epithelial cells as possible substitutes for ameloblasts during tooth regeneration

The disappearance of ameloblasts in erupted teeth hampers the implementation of tissue engineering‐based tooth regeneration. We aimed at utilizing skin epithelial cells as the appropriate substitute for ameloblasts. The conversion potential of 1 day postnatal rat skin epithelial cells to ameloblasts was investigated under the induction of dental papillae mesenchymal cells (DPMCs). Induction strategies had been designed both in vitro and in vivo. Markers for ameloblasts had been detected in skin epithelial cells, which showed a columnar appearance with the nuclei located at one side, under indirect co‐culture with DPMCs in vitro. An enamel–dentine‐like and tooth germ‐like structure was formed by recombining skin epithelial pieces or cells with DPMCs after 14 days of implantation in rat renal capsule. Immunohistochemistry and cell labelling analysis further demonstrated that the enamel‐forming cells were skin epithelium‐derived. These results indicated that the skin epithelium‐derived cells from postnatal rats have the potential to convert to functional ameloblasts under effective induction. Copyright © 2012 John Wiley & Sons, Ltd.     

Immunomodulatory effect of human bone marrow‐derived mesenchymal stromal/stem cells on peripheral blood T cells from rheumatoid arthritis patients

Rheumatoid arthritis (RA) is a Th1/Th17‐mediated autoimmune disease whose current treatment, consisting in the blockage of inflammatory cytokines by disease‐modifying antirheumatic drugs, is not effective for all patients. The therapeutic potential of mesenchymal stromal/stem cells' (MSCs) immunomodulatory properties is being explored in RA. Here, we investigate the effect of human bone marrow (BM)‐MSCs on the expression of cytokines involved in RA physiopathology by the distinct functional compartments of CD4⁺ and CD8⁺ T cells from RA patients. Peripheral blood mononuclear cells from healthy individuals (n = 6) and RA patients (n = 12) were stimulated with phorbol myristate acetate plus ionomycin and cultured in the presence/absence of BM‐MSCs. The expression of (interleukin) IL‐2, tumor necrosis factor alpha (TNF‐α), and interferon‐gamma (IFN‐γ) was evaluated in naive, central memory, effector memory, and effector CD4⁺ and CD8⁺ T cells, whereas IL‐6, IL‐9, and IL‐17 expression was measured in total CD4⁺ and CD8⁺ T cells. mRNA expression of IL‐4, IL‐10, transforming growth factor beta (TGF‐β), cytotoxic T‐lymphocyte‐associated antigen 4, and/or forkhead box P3 was quantified in fluorescence‐activated cell sorting‐purified CD4⁺ T cells, CD8⁺ T cells, and CD4⁺ Treg. BM‐MSCs inhibited the production of TNF‐α, IL‐17, IL‐6, IL‐2, IFN‐γ, and IL‐9 by T cells from RA patients, mainly by reducing the percentage of cells producing cytokines. This inhibitory effect was transversal to all T cell subsets analyzed. At mRNA level, BM‐MSCs increased expression of IL‐10 and TGF‐β by CD4⁺ and CD8⁺ T cells. BM‐MSCs displayed a striking inhibitory action over T cells from RA patients, reducing the expression of cytokines involved in RA physiopathology. Remarkably, BM‐MSC‐derived immunomodulation affected either naive, effector, and memory T cells.     

羊膜上皮细胞和羊膜间充质干细胞构建组织工程皮肤

背景：由于人胎盘来源的间充质干细胞具有多方面的优点,近年来已成为干细胞研究的热点。目的：分析鉴定羊膜间充质干细胞和羊膜上皮细胞的生物学特性,探讨其作为皮肤种子细胞在三维气液培养构建组织工程皮肤中的应用情况。方法：用胰酶胶原酶多步消化法获取羊膜间充质干细胞和羊膜上皮细胞,通过流式细胞术、反转录-聚合酶链反应和免疫荧光染色技术,鉴定两种细胞的表面分子标记、干细胞特性、与皮肤角质形成细胞的相似性,并利用两种细胞为种子细胞以鼠Ⅰ型胶原为基质进行三维气液培养。结果与结论：①流式细胞术检测体外培养羊膜间充质干细胞和羊膜上皮细胞均高表达CD90、CD73、CDl05,不表达造血干细胞标志CD34以及MHC-Ⅱ类分子HLA-DR。②反转录聚合酶链反应检测到羊膜间充质干细胞表达干细胞特性基因CMCY和NANOG,羊膜上皮细胞表达干细胞特性基因CMCY和KLF4,两种细胞均有干细胞特性。③反转录-聚合酶链反应检测羊膜间充质干细胞表达皮肤角质形成细胞特性基因K19、β1-integrin、K8,羊膜上皮细胞表达K19、β1-integrin、K5、K8,免疫荧光染色见羊膜上皮细胞表达与角质形成细胞增殖相关的的特性蛋白K14,说明羊膜上皮细胞与皮肤角质形成细胞更具相似性,在特定条件下更易于分化为皮肤角质形成细胞。④利用两种细胞成功构建组织工程皮肤,苏木精-伊红染色切片显示其具有一定的皮肤结构,且羊膜上皮细胞发生了初步分化。以上结果说明羊膜间充质干细胞与羊膜上皮细胞通过三维培养构建人皮肤组织是可行的。     

Comparison of different culture conditions for human mesenchymal stromal cells for clinical stem cell therapy

Objective. Mesenchymal stromal cells (MSCs) from adult bone marrow (BM) are considered potential candidates for therapeutic neovascularization in cardiovascular disease. When implementing results from animal trials in clinical treatment, it is essential to isolate and expand the MSCs under conditions following good manufacturing practice (GMP). The aims of the study were first to establish culture conditions following GMP quality demands for human MSC expansion and differentiation for use in clinical trials, and second to compare these MSCs with MSCs derived from culture in four media commonly used for MSC cultivation in animal studies simulating clinical stem cell therapy. Material and methods. Human mononuclear cells (MNCs) were isolated from BM aspirates by density gradient centrifugation and cultivated in a GMP‐accepted medium (EMEA medium) or in one of four other media. Results. FACS analysis showed that the plastic‐adherent MSCs cultured in EMEA medium or in the other four media were identically negative for the haematopoietic surface markers CD45 and CD34 and positive for CD105, CD73, CD90, CD166 and CD13, which in combined expression is characteristic of MSCs. MSC stimulation with vascular endothelial growth factor (VEGF) increased expression of the characteristic endothelial genes KDR and von Willebrand factor; the von Willebrand factor and CD31 at protein level as well as the capacity to develop capillary‐like structures. Conclusions. We established culture conditions with a GMP compliant medium for MSC cultivation, expansion and differentiation. The expanded and differentiated MSCs can be used in autologous mesenchymal stromal cell therapy in patients with ischaemic heart disease.     

Vitrification and storage of oral mucosa epithelial cell sheets

Shipping time and shipping delays might affect the quality of the stem cells based engineered “organs.” In our laboratory, we have developed a limbal stem cell deficient (LSCD) rabbit model. To reverse the LSCD, we cultured oral mucosal epithelial cells for 2–3 weeks and engineered cultured autologous oral mucosa epithelial cell sheets (CAOMECS), which were grafted on the LSCD cornea. The purpose of this study was to vitrify CAOMECS and to store it until the CAOMECS can be grafted onto patients. CAOMECS were vitrified in LN₂ for up to 204 days. We tested two different methods of vitrification with different solutions; however, CAOMECS were only viable when they were not stored in a vitrification solution; results were only reported from this CAOMECS. On the basis of hematoxylin and eosin staining, we showed that the CAOMECS morphology was well preserved after long‐term storage in LN₂. Most of the preservation solutions maintained the CAOMECS phenotype (Ki67, proliferating cell nuclear antigen (PCNA), Beta‐Catenin, ZO‐1, E‐Cadherin, CK3, CK4, CK13). The exception was the solution composed with ethylene glycol and Dimethyl sulfoxide (DMSO): this resulted in loss of DeltaN‐p63 expression. DeltaN‐p63 is an important marker for cell proliferation. The expression of proteins involved in cell–cell connection and the differentiation markers were maintained. Apoptosis was not detected in the thawed CAOMECS. We demonstrated that CAOMECS can be stored long‐term in LN₂ without affecting their morphology and phenotype.     

Mesenchymal progenitor cells derived from traumatized human muscle

Mesenchymal stem cells (MSCs) derived from adult tissues are an important candidate cell type for cell‐based tissue engineering and regenerative medicine. Currently, clinical applications for MSCs require additional surgical procedures to harvest the autologous MSCs (i.e. from bone marrow) or commercial allogeneic alternatives. We have recently identified a population of mesenchymal progenitor cells (MPCs) in traumatized muscle tissue that has been surgically debrided from traumatic orthopaedic extremity wounds. The purpose of this study was to evaluate whether MPCs derived from traumatized muscle may provide a clinical alternative to bone‐marrow MSCs, by comparing their morphology, proliferation capacity, cell surface epitope profile and differentiation capacity. After digesting the muscle tissue with collagenase, the MPCs were enriched by a direct plating technique. The morphology and proliferation rate of the muscle‐derived MPCs was similar to bone‐marrow derived MSCs. Both populations expressed cell surface markers characteristic for MSCs (CD 73, CD 90 and CD105), and did not express markers typically absent on MSCs (CD14, CD34 and CD45). After 21 days in specific differentiation media, the histological staining and gene expression of the MPCs and MSCs was characteristic for differentiation into osteoblasts, chondrocytes and adipocytes, but not into myoblasts. Our findings demonstrate that traumatized muscle‐derived MPCs exhibit a similar phenotype and resemble MSCs derived from the bone marrow. MPCs harvested from traumatized muscle tissue may be considered for applications in tissue engineering and regenerative medicine following orthopaedic trauma requiring circumferential debridement. Copyright © 2009 John Wiley & Sons, Ltd.     

Mesenchymal stem cells from rat olfactory bulbs can differentiate into cells with cardiomyocyte characteristics

Mesenchymal stromal/stem cells (MSCs) are widely distributed in different tissues such as bone marrow, adipose tissues, peripheral blood, umbilical cord and amnionic fluid. Recently, MSC‐like cells were also found to exist in rat olfactory bulb and are capable of inducing differentiation into mesenchymal lineages – osteocytes, chondrocytes and adipocytes. However, whether these cells can differentiate into myocardial cells is not known. In this study, we examined whether olfactory bulb‐derived MSCs could differentiate into myocardial cells in vitro. Fibroblast‐like cells isolated from the olfactory bulb of neonatal rats were grown under four conditions: no treatment; in the presence of growth factors (neuregulin‐1, bFGF and forskolin); co‐cultured with cardiomyocytes; and co‐cultured with cardiomyocytes plus neuregulin‐1, bFGF and forskolin. Cell differentiation into myocardial cells was monitored by RT–PCR, light microscopy immunofluorescence, western blot analysis and contractile response to pharmacological treatments. The isolated olfactory bulb‐derived fibroblast‐like cells expressed CD29, CD44, CD90, CD105, CD166 but not CD34 and CD45, consistent with the characteristics of MSCs. Long cylindical cells that spontaneously contracted were only observed following 7 days of co‐culture of MSCs with rat cardiomyocytes plus neuregulin‐1, bFGF and forskolin. RT–PCR and western blot analysis indicated that the cylindrical cells expressed myocardial markers, such as Nkx2.5, GATA4, sarcomeric α‐actinin, cardiac troponin I, cardiac myosin heavy chain, atrial natriuretic peptide and connexin 43. They also contained sarcomeres and gap junction and were sensitive to pharmacological treatments (adrenal and cholinergic agonists and antagonists). These findings indicate that rat olfactory bulb‐derived fibroblast‐like cells with MSC characteristics can differentiate into myocardial‐like cells. Copyright © 2013 John Wiley & Sons, Ltd.     

基底样乳腺癌与管腔A乳腺癌干／祖细胞的表型分析

目的比较人基底样乳腺癌和管腔A乳腺癌干／祖细胞分化前后的细胞表型变化。方法应用乳腺癌细胞球无血清悬浮培养法获取乳腺癌干／祖细胞;分别以荧光免疫技术和流式细胞术检测基底样乳腺癌和管腔A乳腺癌干／祖细胞分化前后细胞角蛋白CK14、CK18、CK19和CD44、CD24的表达情况;分析原代细胞内CD44＋ CD24-细胞水平与克隆形成率的关系。结果基底样乳腺癌干／祖细胞表达CK19＋／CK14＋,不表达CK18,经血清诱导分化后,管腔上皮标志CK18和肌上皮标志CK14呈阳性表达;管腔A乳腺癌干／祖细胞呈CK18＋／CK19＋／CK14-表型,诱导分化后管腔上皮标志CK18和CK19表达,而肌上皮细胞标志CK14则不表达;CD24在基底样乳腺癌表达极低或无表达,但在管腔A乳腺癌高表达。基底样乳腺癌原代细胞中水平较高的是CD44-／CD24-/Low细胞,含CD44＋／CD24-／Low细胞（2．52±0．58）％,而管腔A乳腺癌CD44-／CD24＋细胞所占的比例最高,但不合CD44＋／CD24 -/Low细胞或水平极低。结论基底样乳腺癌和管腔A乳腺癌干／祖细胞有不同的细胞表型,二者的分化能力和分化方向也不同,可能起源于不同的乳腺上皮细胞。     

In vitro and in vivo proliferation,differentiation and migration of cardiac endothelial progenitor cells (SCA1+/CD31+ side‐population cells)

To cite this article: Liang SX, Khachigian LM, Ahmadi Z, Yang M, Liu S, Chong BH. In vitro and in vivo proliferation, differentiation and migration of cardiac endothelial progenitor cells (SCA1⁺/CD31⁺ side‐population cells). J Thromb Haemost 2011; 9 : 1628–37. Summary. Background: Side‐population (SP) cells are a select population identified by a capacity to efflux Hoechst dye and are enriched for stem/progenitor cell activity. Previous studies suggested that cardiac SP (CSP) cells could be divided into SCA1⁺/CD31^? and SCA1⁺/CD31⁺ CSP cells. SCA1⁺/CD31^? CSP cells have been shown to be cardiac stem/progenitor cells. However, SCA1⁺/CD31⁺ CSP cells have not been fully characterized. Objective: The aim of the present study was to characterize SCA1⁺/CD31⁺ CSP cells in the adult mouse heart, and investigate their abilities to proliferate, differentiate, vascularize and migrate in vitro and in vivo. Results: Using fluorescence‐activated cell sorting (FACS), RT‐PCR, and assays of cell proliferation, differentiation and migration, and a murine model of myocardial infarction (MI), we showed that SCA1⁺/CD31⁺ CSP cells express stem cell and endothelial‐specific genes, and reside in the blood vessels. These cells were able to proliferate, differentiate, migrate and vascularize in vitro and in vivo. After MI, SDF‐1α and CXCR4 were up‐regulated in the damaged myocardium and on SCA1⁺/CD31⁺ CSP cells, respectively. Our results further showed that SDF‐1α induced migration of these cells in vitro. Importantly, we found that SCA1⁺/CD31⁺ CSP cells could migrate into the ischemic region from the non‐ischemic area within the myocardium and form a vascular tube‐like structure after MI. Conclusions: Based on the gene expression profile, localization of SCA1⁺/CD31⁺ CSP cells, and their ability to proliferate, differentiate, migrate and vascularize in vitro and in vivo, we postulate that SCA1⁺/CD31⁺ CSP cells may represent endothelial progenitor cells in the mouse heart.     

Interactive endothelial phenotype maintenance and osteogenic differentiation of adipose tissue stromal vascular fraction SSEA‐4+‐derived cells

Bone formation relies on complex processes that require well‐orchestrated interactions between several cell types, such as bone‐forming cells (osteoblasts, OBs) and endothelial cells (ECs). Their co‐culture has been proved relevant to mimicking specific features of the bone niche. Here we propose the co‐culture of microvascular‐like ECs and pre‐OBs, both derived from the SSEA‐4⁺ cell subpopulation from the stromal vascular fraction of human adipose tissue (SSEA‐4⁺ hASCs), to define the conditions in which cells synergistically communicate to support the full differentiation of pre‐OBs and maintenance of the EC phenotype. Co‐cultures of different ratios of the two cell types were established and maintained for up to 21 days in standard endothelial maintenance (ENDO) and osteogenic differentiation (OST) media, as well as in a mixture of these (MIX). The osteogenic maturation of pre‐OBs (ALP activity, OPN and OCN expression, calcium deposition), the evolution of EC numbers (CD31⁺ cells) and maintenance of the endothelial phenotype (CD31 and vWF expression, LDL uptake) were assessed throughout the culture time as a function of cell ratio and culture media. The results obtained demonstrate that EC number has a significant effect on the osteogenic differentiation of pre‐OBs, depending on the medium used. While in ENDO medium the osteogenic differentiation was not observed, in the OST and MIX media it was attained at similar levels, except for the co‐culture with a higher number of ECs in MIX medium. These findings demonstrate that the use of SSEA‐4⁺ hASCs as a single‐cell source is promising to attain 3D bone‐like models with the potential to promote vascularized bone tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd.     

Combined implantation of CD34+ and CD14+ cells increases neovascularization through amplified paracrine signalling

Cell therapy strategies that use adult peripheral blood‐derived CD34⁺ progenitor cells are hampered by low cell numbers and the infrequent cellular incorporation into the neovasculature. Hence, the use of CD34⁺ cells to treat ischaemic diseases is under debate. Interaction between CD34⁺ cells and CD14⁺ cells results in superior endothelial differentiation of CD14⁺ cells in vitro, indicating that cell therapy approaches utilizing both CD34⁺ and CD14⁺ cells may be advantageous in therapeutic neovascularization. Here, human CD34⁺ and CD14⁺ cells were isolated from adult peripheral blood and implanted subcutaneously into nude mice, using matrigel as the carrier. Combined implantation of human CD34⁺ and CD14⁺ cells resulted in superior neovascularization, compared to either cell type alone, albeit incorporation of human cells into the murine vasculature was not observed. Human CD34⁺ and CD14⁺ cells produced and secreted a pentad of pro‐angiogenic mediators, such as HGF, MCP‐1 and IL‐8, bFGF and VEGFa in monoculture. The production and secretion of pro‐angiogenic mediators by CD14⁺ cells was highly amplified upon incubation with conditioned medium from CD34⁺ cells. In vivo, neovascularization of matrigel implants did not rely on the endothelial differentiation and incorporation of CD34⁺ or CD14⁺ cells, but depended on the paracrine effects of IL‐8, MCP‐1, HGF, bFGF and VEGFa secreted by implanted cells. Administration of this growth factor/cytokine pentad using matrigel as a carrier results in cell recruitment and microvessel formation equal to progenitor cell‐induced neovascularization. These data provide new insights on neovascularization by cell therapy and may contribute to new strategies for the treatment of ischaemic diseases. Copyright © 2011 John Wiley & Sons, Ltd.     

Cytokeratin 19 fragments in patients with acute lung injury: a preliminary observation

Objective Cytokeratin 19 (CK19) is a specific cytoskeletal structure for alveolar epithelium. We hypothesized that the levels of CK19 fragments in bronchoalveolar lavage (BAL) fluid could serve as an index of epithelial injury and as a prognosis marker in patients with acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). The aims of our study were, in patients with ALI/ARDS: (1) to measure CK19 fragments concentrations in BAL fluid, (2) to assess its prognostic value, and (3) to identify the cellular source of CK19 in the alveolar space.Design Prospective preliminary study.Setting University hospital surgical ICU.Patients Twenty-two mechanically ventilated patients with ALI/ARDS and 10 non-ventilated control patients. Plasma samples were obtained for 11 ALI/ARDS patients.Measurements and results The concentration of BAL CK19 fragments was higher in patients (median 4916 pg/ml, 25th–75th percentile 2717–10533) than in controls (2208 pg/ml. 767–3923; p = 0.05), and higher in 10 non-survivors (7051 pg/ml, 4372–13371) than in 12 survivors (2888 pg/ml, 1315–5639; p = 0.03 among ALI/ARDS patients). BAL CK19 fragment concentration did not correlate with simplified acute physiologic score, lung injury score or PaO₂/FIO₂ ratio, but correlated positively with BAL albumin concentration (p = 0.002) and with number of BAL macrophages (p = 0.0001). Plasma CK19 fragment concentrations were 10 times lower than those in BAL. Immunohistochemical staining for CK19 showed a strong labelling of injured detached epithelial cells and hyperplastic epithelium in ALI/ARDS lung samples.Conclusion CK19 fragment concentrations were found to be elevated in BAL fluid in ALI/ARDS patients compared with control subjects. High BAL CK19 fragment levels were associated with a poor prognosis.Electronic supplementary material The electronic reference of this article is . The online full-text version of this article includes electronic supplementary material. This material is available to authorised users and can be accessed by means of the ESM button beneath the abstract or in the structured full-text article. To cite or link to this article you can use the above reference.     

New insights into epithelial differentiation of human adipose‐derived stem cells

Although many studies using stem cells as therapeutic agents after renal failure have been published in recent years, our knowledge of the factors involved and the cellular mechanisms underlying their beneficial effect on organ regeneration is incomplete. A growing insight into these interactions would help to utilize the biological potential of stem cells for therapeutic approaches. It is here hypothesized that soluble factors released by tubular epithelial cells (TECs) induce epithelial differentiation in adipose‐derived adult mesenchymal stem cells (ASCs). ASCs were therefore cultured in conditioned medium (CM) derived from TECs and the changes in expression genes towards an epithelial pattern were determined by microarray and qPCR analyses. The changes in gene expression were evaluated using Affymetrix HG‐U133 Plus 2.0 arrays. Microarray‐based screening revealed 117 genes differentially expressed in a significant manner after short‐time incubation (3 days) of ASCs with CM, and four of these were solute carriers (SLCs). Changes in mRNA expression of these SLCs were verified by qPCR at several time points, additionally with four stem cell factors and five epithelial markers. qPCR analyses showed that expression of three of the SLCs rose significantly, whereas three of the four stem cell markers analysed decreased during 7 days of CM incubation. Moreover, a robust expression of three characteristic epithelial markers (cytokeratin 18, ZO‐1 and ZO‐2) was observed after 17 days. These changes in the expression patterns strongly indicate differentiation towards the epithelial lineage. The capability of ASCs to differentiate into epithelial cells may be important in organ repair mechanisms. Copyright © 2011 John Wiley & Sons, Ltd.     

Effects of resveratrol on enrichment of adipose‐derived stem cells and their differentiation to osteoblasts in two‐and three‐dimensional cultures

The goal of this study was to develop a method for increasing the yield of multipotent adipose‐derived mesenchymal stem cells (ASCs) and osteoprogenitor cells (OPCs) from subcutaneous fat. After removing mature adipocytes and haematopoietic cells from rat inguinal fat, ASCs in the remaining cell population were verified by their attachment to plastic, surface marker profile (CD271⁺, CD73⁺ and CD45^–) and ability to differentiate into adipocytes, chondrocytes and osteoblasts. OPCs were defined as E11⁺ and OCN⁺. Adherent cells were cultured in growth medium (GM) or osteogenic medium (OM) and treated with resveratrol (0, 12.5, and 25 µ m ) for 7 days; ASCs and OPCs were assessed by flow cytometry. Osteogenic potential was determined in two‐dimensional (2D) cultures as a function of alkaline phosphatase‐specific activity and osteocalcin production. In addition, cells were seeded onto three‐dimensional (3D) poly‐ε‐caprolactone scaffolds and cultured under dynamic conditions; mineralization was quantified by micro‐CT at 4, 8 and 12 weeks. Resveratrol increased the percentage of ASCs in the population (population%) and number of ASCs in both GM and OM, but increased only the number of OPCs in GM. In both media types resveratrol increased alkaline phosphatase activity and osteocalcin levels. In 3D cultures, resveratrol‐treated cells significantly increased mineralized matrix volume at early time points. Resveratrol exerted a biphasic effect on adherent cells by enriching the ASC and OPC populations and enhancing osteogenic differentiation. Resveratrol pretreatment induced more mineralization at earlier time points and represents a clinically viable technique for orthopaedic and dental applications for autologous stem cell therapy. Copyright © 2012 John Wiley & Sons, Ltd.