Cardiac potential of stem cells from whole human umbilical cord tissue

We investigated the role of stem cells from human umbilical cord tissue in cardiomyocyte regeneration. The umbilical cord stem cells were initially characterized and differentiated in a myocardial differentiation medium containing 5‐azacytidine for 24 h. Differentiation into cardiomyocytes was determined by expression of cardiac specific markers, like cardiac α‐actin, connexin43, myosin, Troponin T, and ultrastructural analysis. In vivo, the transplanted umbilical cord stem cells were sprouting from local injection and differentiated into cardiomyocyte‐like cells in a rat myocardial infarction model. Echocardiography revealed increasing left ventricular function after umbilical cord stem cell transplantation. These results demonstrate that umbilical cord stem cells can differentiate into cardiomyocyte‐like cells both in vitro and in vivo. Therefore, human umbilical cord might represent a source of stem cells useful for cellular therapy and myocardial tissue engineering. Future studies are required to determine the molecular signaling mechanisms responsible for this phenomenon. J. Cell. Biochem. 107: 926–932, 2009. © 2009 Wiley‐Liss, Inc.     

The similar effect of transplantation of marrow-derived mesenchymal stem cells with or without prior differentiation induction in experimental myocardial infarction

Marrow-derived mesenchymal stem cells (MSCs) have been heralded as a source of great promise for the regeneration of the infarcted heart. There is no clear data indicating whether or not in vitro differentiation of MSCs into major myocardial cells can increase the beneficial effects of MSCs. The aim of this study is to address this issue. To induce MSCs to transdifferentiate into cardiomyocyte-like and endothelial-like cells, 5-azacytidine and vascular endothelial growth factor (VEGF) were used, respectively. Myocardial infarction in rabbits was generated by ligating the left anterior descending coronary artery. Animals were divided into three experimental groups: I, control group; II, undifferentiated mesenchymal stem cell transplantation group; III, differentiated mesenchymal stem cell transplantation group; which respectively received peri-infarct injections of culture media, autologous undifferentiated MSCs and autologous differentiated MSCs. General pathology, immunohistochemistry, electron microscopy and echocardiography were performed in order to search for myocardial regeneration and improvement of cardiac function. In Groups II and III, implanted cells transdifferentiate into myocardial cells within 28 days post injection in a similar manner, and well-developed ultra structures formed within transplanted cells. Improvements in left ventricular function and reductions in infarcted area were observed in both cell-transplanted groups to the same degree. Vascular density was similar in Groups II and III and significantly higher in these groups compared with the control group. There is no need for prior differentiation induction of marrow-derived MSCs before transplantation and peri-infarct implantation of MSCs can efficiently regenerate the infarcted myocardium and improve cardiac function.     

Granulocyte‐Macrophage Colony‐Stimulating Factor (GM‐CSF) Controls the Proliferation and Differentiation of Mouse Epidermal Melanocytes from Pigmented Spots Induced by Ultraviolet Radiation B

Repeated exposure of ultraviolet radiation B (UVB) on the dorsal skin of hairless mice induces the development of pigmented spots long after its cessation. The proliferation and differentiation of epidermal melanocytes in UVB‐induced pigmented spots are greatly increased, and those effects are regulated by keratinocytes rather than by melanocytes. However, it remains to be resolved what factor(s) derived from keratinocytes are involved in regulating the proliferation and differentiation of epidermal melanocytes. In this study, primary melanoblasts (c. 80%) and melanocytes (c. 20%) derived from epidermal cell suspensions of mouse skin were cultured in a basic fibroblast growth factor‐free medium supplemented with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). GM‐CSF induced the proliferation and differentiation of melanocytes in those keratinocyte‐depleted cultures. Moreover, an antibody to GM‐CSF inhibited the proliferation of melanoblasts and melanocytes from epidermal cell suspensions derived from the pigmented spots of UV‐irradiated mice, but not from control mice. Further, the GM‐CSF antibody inhibited the proliferation and differentiation of melanocytes co‐cultured with keratinocytes derived from UV‐irradiated mice, but not from control mice. The quantity of GM‐CSF secreted from keratinocytes derived from the pigmented spots of UV‐irradiated mice was much greater than that secreted from keratinocytes derived from control mice. Moreover, immunohistochemistry revealed the expression of GM‐CSF in keratinocytes derived from the pigmented spots of skin in UV‐irradiated mice, but not from normal skin in control mice. These results suggest that GM‐CSF is one of the keratinocyte‐derived factors involved in regulating the proliferation and differentiation of mouse epidermal melanocytes from UVB‐induced pigmented spots.     

Matrix stiffness modulates the differentiation of neural crest stem cells in vivo

Stem cells are often transplanted with scaffolds for tissue regeneration; however, how the mechanical property of a scaffold modulates stem cell fate in vivo is not well understood. Here we investigated how matrix stiffness modulates stem cell differentiation in a model of vascular graft transplantation. Multipotent neural crest stem cells (NCSCs) were differentiated from induced pluripotent stem cells, embedded in the hydrogel on the outer surface of nanofibrous polymer grafts, and implanted into rat carotid arteries by anastomosis. After 3 months, NCSCs differentiated into smooth muscle cells (SMCs) near the outer surface of the polymer grafts; in contrast, NCSCs differentiated into glial cells in the most part of the hydrogel. Atomic force microscopy demonstrated a stiffer matrix near the polymer surface but much lower stiffness away from the polymer graft. Consistently, in vitro studies confirmed that stiff surface induced SMC genes whereas soft surface induced glial genes. These results suggest that the scaffold’s mechanical properties play an important role in directing stem cell differentiation in vivo, which has important implications in biomaterials design for stem cell delivery and tissue engineering.     

Heparan sulfate maintains adult midgut homeostasis in Drosophila

Tissue homeostasis is controlled by the differentiated progeny of residential progenitors (stem cells). Adult stem cells constantly adjust their proliferation/differentiation rates to respond to tissue damage and stresses. However, how differentiated cells maintain tissue homeostasis remains unclear. Here, we find that heparan sulfate (HS), a class of glycosaminoglycan (GAG) chains, protects differentiated cells from loss to maintain intestinal homeostasis. HS depletion in enterocytes (ECs) leads to intestinal homeostasis disruption, with accumulation of intestinal stem cell (ISC)‐like cells and mis‐differentiated progeny. HS‐deficient ECs are prone to cell death/stress and induced cytokine and epidermal growth factor (EGF) expression, which, in turn, promote ISC proliferation and differentiation. Interestingly, HS depletion in ECs results in the inactivation of decapentaplegic (Dpp) signaling. Moreover, ectopic Dpp signaling completely rescued the defects caused by HS depletion. Together, our data demonstrate that HS is required for Dpp signal activation in ECs, thereby protecting ECs from ablation to maintain midgut homeostasis. Our data shed light into the regulatory mechanisms of how differentiated cells contribute to tissue homeostasis maintenance.     

Characterization of hematopoietic potential of mesenchymal stem cells

Mesenchymal and hematopoietic tissues are important reservoirs of adult stem cells. The potential of tissue resident mesenchymal stem cells (MSCs) to differentiate into cells of mesodermal and ectodermal lineages has been reported previously. We examined the hypothesis that adherent adipose tissue resident mesenchymal stem cells (ASCs) are capable of generating cells with hematopoietic characteristics. When cultured in differentiation media, clonally isolated ASCs develop into cells with hematopoietic attributes. The hematopoietic differentiated cells (HD) express early hematopoietic (c‐kit, PROM1, CD4) as well as monocyte/macrophage markers (CCR5, CD68, MRC1, CD11b, CSF1R). Additionally, HD cells display functional characteristics of monocyte/macrophages such as phagocytosis and enzymatic activity of α‐Naphthyl Acetate Esterase. HD cells are also responsive to stimulation by IL‐4 and LPS as shown by increased CD14 and HLA‐DRB1 expressions and release of IL‐2, IL10, and TNF. Taken together, this study characterizes the potential of ASCs to generate functional macrophages in vitro, and therefore paves way for their possible use in cell therapy applications. J. Cell. Physiol. 225: 888–897, 2010. © 2010 Wiley‐Liss, Inc.     

Mesenchymal stem cells: clinical applications and biological characterization

Mesenchymal stem cells (MSCs) have been isolated from bone marrow, periosteum, trabecular bone, adipose tissue, synovium, skeletal muscle and deciduous teeth. These cells have the capacity to differentiate into cells of connective tissue lineages, including bone, fat, cartilage and muscle. A great deal has been learned in recent years about the isolation and characterization of MSCs, and control of their differentiation. These cells have generated a great deal of interest because of their potential use in regenerative medicine and tissue engineering and there are some dramatic examples, derived from both pre-clinical and clinical studies, that illustrate their therapeutic value. This review summarizes recent findings regarding the potential clinical use of MSCs in cardiovascular, neural and orthopaedic applications. As new methods are developed, there are several aspects to the implanted cell-host interaction that need to be addressed before we can fully understand the underlying mechanisms. These include the host immune response to implanted cells, the homing mechanisms that guide delivered cells to a site of injury and the differentiation in vivo of implanted cells under the influence of local signals.     

Adipose-derived adult stem cells for cartilage tissue engineering

Tissue engineering is a promising therapeutic approach that uses combinations of implanted cells, biomaterial scaffolds, and biologically active molecules to repair or regenerate damaged or diseased tissues. Many diverse and increasingly complex approaches are being developed to repair articular cartilage, with the underlying premise that cells introduced exogenously play a necessary role in the success of engineered tissue replacements. A major consideration that remains in this field is the identification and characterization of appropriate sources of cells for tissue-engineered repair of cartilage. In particular, there has been significant emphasis on the use of undifferentiated progenitor cells, or "stem" cells that can be expanded in culture and differentiated into a variety of different cell types. Recent studies have identified the presence of an abundant source of stem cells in subcutaneous adipose tissue. These cells, termed adipose-derived adult stem (ADAS) cells, show characteristics of multipotent adult stem cells, similar to those of bone marrow derived mesenchymal stem cells (MSCs), and under appropriate culture conditions, synthesize cartilage-specific matrix proteins that are assembled in a cartilaginous extracellular matrix. The growth and chondrogenic differentiation of ADAS cells is strongly influenced by factors in the biochemical as well as biophysical environment of the cells. Furthermore, there is strong evidence that the interaction between the cells, the extracellular biomaterial substrate, and growth factors regulate ADAS cell differentiation and tissue growth. Overall, ADAS cells show significant promise for the development of functional tissue replacements for various tissues of the musculoskeletal system.     

Transplantation of stem/progenitor cells of human brain into the brain of adult rats

We studied the development of stem/progenitor cells of the human brain transplanted in the adult rat brain after reproduction in an in vitro tissue culture. It was preliminarily shown by the immunological methods that the stem cells grown in a medium with growth factors formed neurospheres, which were heterogenous and contained both stem and progenitor cells of the human brain. The cells were implanted in the hippocampus, striatum, or lateral ventricle of the rat brain as a suspension or aggregates (neurospheres) and their behavior and differentiation were studies within 10, 20, and 30 days using the morphological and immunochemical methods. The cultured cells of the human brain continued their development in the rat brain, migrated, and formed neurons and astrocytes. The white mater fibers, lateral ventricle wall, and perivascular spaces served as the main pathways of migration. The neuronal differentiation was shown by staining with antibodies to beta-tubulin III, neurofilaments-70, and calbindin. Some growing nerve cells had long processes with growth cones. At the same time, some transplanted cells retained the undifferentiated state within one month after the implantation, as shown by the vimentin expression.     

Study of differentiated human umbilical cord–derived mesenchymal stem cells transplantation on rat model of advanced parkinsonism

The aim of this study was to explore the curative effect of differentiated human umbilical cord–derived mesenchymal stem cells (hUC‐MSCs) transplantation on rat of advanced Parkinson disease (PD) model. Human umbilical cord–derived mesenchymal stem cells were cultured and induced differentiation in vitro. The PD rats were established and allocated randomly into 2 groups: differentiated hUC‐MSCs groups and physiological saline groups (the control group). Rotation test and immunofluorescence double staining were done. The result showed that hUC‐MSCs could differentiate into mature dopamine neurons. Frequency of rotation was significantly less in differentiated hUC‐MSCs groups than in normal saline group. After we transplanted these cells into the unilateral lesioned substantia nigra induced by striatal injection of 6‐hydroxydopamine and performed in the medial forebrain bundle and ventral tegmental area, nigral tyrosine hydroxylase–positive cells were observed and survival of at least 2 months. In addition, transplantation of hUC‐MSCs could make an obviously therapeutic effect on PD rats.     

Effect of preventive and regressive isosorbide 5-mononitrate treatment on catecholamine levels in plasma, platelets, adrenals, left ventricle and aorta in cyclosporin A-induced hypertensive rats

Increased vascular reactivity associated with cyclosporin A (CsA)-induced arterial hypertension might result from increased vasoconstriction and/or decreased vasodilatation. The administration of organic NO donors could have beneficial effects by the NO-cGMP reposition, but there is the risk of sympathetic nervous system worsening by neuro-hormonal counter-regulation. We evaluate the effect of preventive and regressive (curative) isosorbide 5-mononitrate (Is-5-Mn) treatment on blood pressures and on plasma, platelets, adrenals, left ventricle and aorta norepinephrine (NE) and epinephrine (E) contents, assessed by HPLC, in CsA-induced hypertensive rats. Five rat groups were tested: control (orange juice), CsA (5 mg/kg/day) and Is-5-Mn (150 mg/kg/day, bid) groups were treated for 7 weeks; preventive group (Is-5-Mn+CsA): Is-5-Mn during 2 weeks plus 7 weeks of Is-5-Mn+CsA; regressive group (CsA+Is-5-Mn): CsA during 7 weeks plus 5 weeks of CsA+Is-5-Mn. The increased BP in the CsA group was prevented, but was not reverted, by concomitant Is-5-Mn treatment. In the CsA-treated rats, there was a noticeable decrease in left ventricle NE and E contents and aorta NE levels and a moderate increase in circulating catecholamines, without significant effect in the adrenals values. When Is-5-Mn was preventively used, the CsA-induced effect on left ventricle and aorta was prevented. Concomitantly, however, the plasma-platelet catecholamine balance was disrupted, accumulating NE in plasma, whereas E increased in aorta, mimic the single Is-5-Mn-treated group. In opposition, in the group used as regressive Is-5-Mn therapy, the adrenals contents were higher compared with the CsA-group and, simultaneously, the CsA-evoked effects on circulating, left ventricle and aorta catecholamines were not reverted. In conclusion, regressive Is-5-Mn therapy was unable to attenuate CsA-induced catecholamine changes and BP values even worsened. On the contrary, preventive Is-5-Mn treatment prevented the catecholamine changes on left ventricle and aorta, but increased plasma NE and aorta E accumulation. Even though with those effects, hypertension development was totally prevented, suggesting that peripheral SNS per se cannot fully explain CsA-induced hypertension. Furthermore, Is-5-Mn might produce beneficial effects only if preventively employed but, considering the changes on peripheral catecholamine contents, a judicious evaluation of the nitrate therapy impact is recommended in order to avoid further deleterious effects.     

Regeneration of infarcted myocardium with resveratrol-modified cardiac stem cells

The major problem in stem cell therapy includes viability and engraftment efficacy of stem cells after transplantation. Indeed, the vast majority of host-transfused cells do not survive beyond 24-72 hrs. To increase the survival and engraftment of implanted cardiac stem cells in the host, we developed a technique of treating these cells with resveratrol, and tested it in a rat model of left anterior descending (LAD) occlusion. Multi-potent clonogenic cardiac stem cells isolated from rat heart and stably transfected with EGFP were pre-treated with 2.5 μM resveratrol for 60 min. Rats were anaesthetized, hearts opened and the LAD occluded to induce heart attack. One week later, the cardiac reduced environment was confirmed in resveratrol treated rat hearts by the enhanced expression of nuclear factor-E2-related factor-2 (Nrf2) and redox effector factor-1 (Ref-1). M-mode echocardiography after stem cell therapy, showed improvement in cardiac function (left ventricular ejection fraction, fractional shortening and cardiac output) in both, the treated and control group after 7 days, but only resveratrol-modified stem cell group revealed improvement in cardiac function at the end of 1, 2 and 4 months time. The improvement of cardiac function was accompanied by enhanced stem cell survival and engraftment as demonstrated by the expression of cell proliferation marker Ki67 and differentiation of stem cells towards the regeneration of the myocardium as demonstrated by the expression of EGFP up to 4 months after LAD occlusion in the resveratrol-treated stem cell group. Expression of stromal cell-derived factor and myosin conclusively demonstrated homing of stem cells in the infarcted myocardium, its regeneration leading to improvement of cardiac function.     

Differential effect of myocardial matrix and integrins on cardiac differentiation of human mesenchymal stem cells

Dysregulation of matrix synthesis during myocardial fibrosis in post-infarct ventricular remodeling contributes to ventricular dysfunction. Bone marrow stem cell transplantation prevents functional deterioration following myocardial infarction. However, effect of myocardial extracellular matrix (ECM) on stem cell differentiation is poorly understood. We investigate the role of collagen matrices and integrin system in cardiac differentiation and engraftment of stem cells in infarcted myocardium. Sternum-derived bone marrow mesenchymal stem cells (MSCs) were differentiated into cardiomyocyte-like cells (CLCs). They were characterized using RT-PCR, immunofluorescence, flow cytometry and functional integrin neutralization assays. CLCs were injected into peri-infarct borders of injured myocardium of Wistar rats one week following left anterior descending (LAD) artery ligation. Cardiac function was analyzed via pressure-volume relationships. Cardiac differentiated CLCs displayed collagen V specificity, which was absent in undifferentiated MSCs. Collagen V, but not collagen I matrix, promoted attachment, proliferation and cardiac differentiation of CLCs. In contrast to β₁, α_v integrin contributed minimally in the attachment of CLCs on collagen matrices. However, inhibition of α_vβ_3, but not α₂β₁ integrin, selectively attenuated troponin T, sarcomeric α-actin and ryanodine 2 receptor gene expression in CLCs. Both MSC and CLC transplantation prevented chamber dilatation and improved contractile function. However, systolic activity in MSC transplanted animals was accompanied by heightened wall stress as demonstrated by elevated myocardial end-diastolic pressure and prolonged tissue relaxation time. Localization of CLCs in the vicinity of collagen V-expressing myofibers promoted their integration into cardiac syncytium. CLCs may facilitate hemodynamic recovery by preserving tissue elasticity in the peri-infarct borders that sustains contractile efficiency for functional recovery in an actively remodeling infarcted myocardium.     

Adult stem cell homing and differentiation in vitro on composite fibrin matrix

Strategies to generate differentiated cells from haematopoetic progenitor cells will enhance potential use of adult stem cells for therapeutic transplantation or tissue engineering. Transplantation of undifferentiated stem cells into recipient tissue hinges on the hypothesis of a milieu dependent differentiation and it has been suggested that a clot-equivalent scaffold is crucial for these circulating cells to anchor and multiply. Here a natural scaffold, fibrin along with fibronectin, gelatin and growth factors has been used to induce endothelial progenitor cells and smooth muscle progenitor cells to differentiate into endothelial cells and smooth muscle cells, respectively, from peripheral blood mononuclear cells. Characteristics of endothelial cells have been verified by the detection of mRNA for and immunostaining the cells for von Willebrand factor, uptake of acetylated low-density lipoproteins and measurement of released nitric oxide in the culture medium, as nitrite. The specific molecules that characterized smooth muscle cells were alpha smooth muscle actin and calponin, besides deposition of collagen type I and elastin, onto the culture matrix. The adhesive proteins used for the fabrication of endothelial progenitor cells matrix and smooth muscle progenitor cells matrix were the same, but specific differentiation was brought about by modulating the growth factor composition in the matrix and in the culture medium. Both endothelial and smooth muscle cells were consistently developed from 20 ml of human blood.     

Pluripotent mesenchymal stem cells reside within avian connective tissue matrices

Summary Recent studies have noted the presence of putative stem cells derived from the connective tissues associated with skeletal muscle, heart, and dermis. Long-term continuous cultures of these cells from each tissue demonstrated five distinct phenotypes of mesodermal origin, i.e. muscle, fat, cartilage, bone, and connective tissue. Clonal analysis was performed to determine whether these morphologies were the result of a mixed population of lineage-committed stem cells or the differentiation of pluripotent stem cells or both. Putative stem cells from four tissues (skeletal muscle, dermis, atria, and ventricle) were isolated and cloned. Combined, 1158 clones were generated from the initial cloning and two subsequent subclonings. Plating efficiency approximated 5.8%. Approximately 70% of the 1158 clones displayed a pure stellate morphology, while the remaining clones contained a mixture of stellate, chondrogenic- or osteogenic-like morphologies or both. When cultured in the presence of dexamethasone, cells from all clones differentiated in a time- and concentration-dependent manner into muscle, fat, cartilage, and bone. These results suggest that pluripotent mesenchymal stem cells are present within the connective tissues of skeletal muscle, dermis, and heart and may prove useful for studies concerning the regulation of stem cell differentiation, wound healing, and tissue restoration, replacement and repair.     

Optimization of primary culture condition for mesenchymal stem cells derived from umbilical cord blood with factorial design

Mesenchymal stem cells (MSCs) can not only support the expansion of hematopoietic stem cells in vitro, but also alleviate complications and accelerate recovery of hematopoiesis during hematopoietic stem cell transplantation. However, it proved challenging to culture MSCs from umbilical cord blood (UCB) with a success rate of 20–30%. Many cell culture parameters contribute to this outcome and hence optimization of culture conditions is critical to increase the probability of success. In this work, fractional factorial design was applied to study the effect of cell inoculated density, combination and dose of cytokines, and presence of serum and stromal cells. The cultured UCB‐MSC‐like cells were characterized by flow cytometry and their multilineage differentiation potentials were tested. The optimal protocol was identified achieving above 90% successful outcome: 2 × 10⁶ cells/mL mononuclear cells inoculated in Iscove's modified Dulbecco's medium supplied with 10% FBS, 15 ng/mL IL‐3, and 5 ng/mL Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). Moreover, the UCB‐MSC‐like cells expressed MSC surface markers of CD13, CD29, CD105, CD166, and CD44 positively, and CD34, CD45, and human leukocyte antigens‐DR (HLA‐DR) negatively. Meanwhile, these cells could differentiate into osteoblasts, chondrocytes, and adipocytes similarly to MSCs derived from bone marrow. In conclusion, we have developed an efficient protocol for the primary culture of UCB‐MSCs by adding suitable cytokines into the culture system. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Relationship of intracerebral pituitary grafts to central neuropeptide systems

Variable amounts of pituitary tissue from neonatal or 30-day-old donor rats were implanted in the recessus triangularis or third ventricle of hypophysectomized male host rats. The pituitary tissue was implanted either immediately or 30 days after hypophysectomy of the host rat. Grafts from donors of either age were capable of maintaining a significant degree of testicular weight in one-third of the implanted animals. Neonatal grafts were not capable of restoring testicular weight when implanted 30 days after hypophysectomy. Final body weights of all graft-bearing animals were greater than those of hypophysectomized controls. The pars distalis of all grafts contained large numbers of cells immunore-active for LH, GH, and ACTH; TSH-immunoreactive cells were sparse. Prolactin-positive cells were extensive in grafts of animals in which the testes were maintained, and virtually absent in grafts of animals with atrophic testes. The fiber systems of three central neuropeptides, LRF, SRIF, and ACTH, were stained and found not to enter the graft. The results suggest that pituitary grafts in the third ventricle may receive their hypophysiotropic neuropeptides from the CSF.     

Effects of chitosan/collagen substrates on the behavior of rat neural stem cells

Spinal cord and brain injuries usually lead to cavity formation. The transplantation by combining stem cells and tissue engineering scaffolds has the potential to fill the cavities and replace the lost neural cells. Both chitosan and collagen have their unique characteristics. In this study, the effects of chitosan and collagen on the behavior of rat neural stem cells (at the neurosphere level) were tested in vitro in terms of cytotoxicity and supporting ability for stem cell survival, proliferation and differentiation. Under the serum-free condition, both chitosan membranes and collagen gels had low cytotoxicity to neurospheres. That is, cells migrated from neurospheres, and processes extended out from these neurospheres and the differentiated cells. Compared with the above two materials, chitosan-collagen membranes were more suitable for the co-culture with rat neural stem cells, because, except for low cytotoxicity and supporting ability for the cell survival, in this group, a large number of cells were observed to migrate out from neurospheres, and the differentiating percentage from neurospheres into neurons was significantly increased. Further modification of chitosan-collagen membranes may shed light on in vivo nerve regeneration by transplanting neural stem cells.