Human Umbilical Cord-Derived Mesenchymal Stromal Cells Improve Left Ventricular Function, Perfusion, and Remodeling in a Porcine Model of Chronic Myocardial Ischemia.

UNLABELLED: : Stem cell therapy has emerged as a new strategy for treatment of ischemic heart disease. Although umbilical cord-derived mesenchymal stromal cells (UC-MSCs) have been used preferentially in the acute ischemia model, data for the chronic ischemia model are lacking. In this study, we investigated the effect of UC-MSCs originated from Wharton's jelly in the treatment of chronic myocardial ischemia in a porcine model induced by ameroid constrictor. Four weeks after ameroid constrictor placement, the surviving animals were divided randomly into two groups to undergo saline injection (n = 6) or UC-MSC transplantation (n = 6) through the left main coronary artery. Two additional intravenous administrations of UC-MSCs were performed in the following 2 weeks to enhance therapeutic effect. Cardiac function and perfusion were examined just before and at 4 weeks after intracoronary transplantation. The results showed that pigs with UC-MSC transplantation exhibited significantly greater left ventricular ejection fraction compared with control animals (61.3% ± 1.3% vs. 50.3% ± 2.0%, p < .05). The systolic thickening fraction in the infarcted left ventricular wall was also improved (41.2% ± 3.3% vs. 46.2% ± 2.3%, p < .01). Additionally, the administration of UC-MSCs promoted collateral development and myocardial perfusion. The indices of fibrosis and apoptosis were also significantly reduced. Immunofluorescence staining showed clusters of CM-DiI-labeled cells in the border zone, some of which expressed von Willebrand factor. These results suggest that UC-MSC treatment improves left ventricular function, perfusion, and remodeling in a porcine model with chronic myocardial ischemia. SIGNIFICANCE: Ischemic heart disease is the leading cause of death worldwide. Many patients with chronic myocardial ischemia are not suitable for surgery and have no effective drug treatment; they are called "no-option" patients. This study finds that umbilical cord-derived mesenchymal stromal cells transplanted by intracoronary delivery combined with two intravenous administrations was safe and could significantly improve left ventricular function, perfusion, and remodeling in a large-animal model of chronic myocardial ischemia, which provides a new choice for the no-option patients. In addition, this study used clinical-grade mesenchymal stem cells with delivery and assessment methods commonly used clinically to facilitate further clinical transformation.

The spatiotemporal development of intercalated disk in three-dimensional engineered heart tissues based on collagen/matrigel matrix.

Intercalated disk (ID), which electromechanically couples cardiomyocytes into a functional syncitium, is closely related to normal morphology and function of engineered heart tissues (EHTs), but the development mode of ID in the three-dimensional (3D) EHTs is still unclear. In this study, we focused on the spatiotemporal development of the ID in the EHTs constructed by mixing neonatal rat cardiomyocytes with collagen/Matrigel, and investigated the effect of 3D microenvironment provided by collagen/Matrigel matrix on the formation of ID. By histological and immmunofluorescent staining, the spatiotemporal distribution of ID-related junctions was detected. Furthermore, the ultra-structures of the ID in different developmental stages were observed under transmission electron microscope. In addition, the expression of the related proteins was quantitatively analyzed. The results indicate that accompanying the re-organization of cardiomyocytes in collagen/Matrigel matrix, the proteins of adherens junctions, desmosomes and gap junctions redistributed from diffused distribution to intercellular regions to form an integrated ID. The adherens junction and desmosome which are related with mechanical connection appeared earlier than gap junction which is essential for electrochemical coupling. These findings suggest that the 3D microenvironment based on collagen/Matrigel matrix could support the ordered assembly of the ID in EHTs and have implications for comprehending the ordered and coordinated development of ID during the functional organization of EHTs.

[Inhibitory effects of human umbilical cord-derived mesenchymal stem cells on proliferation of peripheral blood mononuclear cells from spondyloarthritis patients].

OBJECTIVE: To explore the inhibitory effects of human umbilical cord-derived mesenchymal stem cells (hUCMSC) on the proliferation of peripheral blood mononuclear cells (PBMC) from spondyloarthritis (SpA) patients. METHODS: A total of 12 SpA patients at Chinese PLA General Hospital were recruited from May 2012 to October 2012. Information on demographic characteristics, disease and functional activity was collected. Isolated PBMC were stimulated by phytohemagglutinin (PHA, 1 µg/ml) in the presence or absence of hUCMSC.The proliferation of hUCMSC was suppressed by irradiation with Co60 (30 Gy) before co-culturing with PBMC. The proliferation of PBMC was determined by Cell Counting Kit-8 (CCK-8). Cell cycle profiles of PBMC were analyzed by flow cytometry. The association of inhibitory effect of hUCMSC with the disease and functional activity of SpA patients was examined. RESULTS: After coculturing with hUCMSC by cell-to-cell contact for 5 days, the proliferation of PBMC stimulated by PHA (1 µg/ml) was significantly inhibited by hUCMSC in a dose-dependent manner.The inhibition rate of the proliferation of PBMC cocultured with hUCMSC by cell-to-cell contact was higher than that by Transwell culture (57% ± 17% vs 32% ± 12%, P < 0.01). Compared to PBMC cultured alone, a larger number of PBMC cocultured with hUCMSC were in phase G1 (86% ± 3% vs 68% ± 5%, P < 0.01) while a lower number of cells in phases S and G2 (8% ± 3% vs 26% ± 5%, P < 0.01). No association was found between the inhibitory effect of hUCMSC and the disease and functional activity. CONCLUSION: The proliferation of PBMC from SpA patients may be inhibited by hUCMSC. And hUCMSC have therapeutic potentials for SpA patients.

[Inhibitory effect of human umbilical cord-derived mesenchymal stem cells on interleukin-17 production in peripheral blood T cells from spondyloarthritis patients].

In this study, the inhibitory effect of human umbilical cord-derived mesenchymal stem cells (hUCMSC) on interleukin-17 (IL-17) production in peripheral blood T cells from patients with spondyloarthritis (SpA) were investigated, in order to explore the therapeutic potential of hUCMSC in the SpA. Peripheral blood mononuclear cells (PBMNC) were isolated from patients with SpA (n = 12) and healthy subjects (n = 6). PBMNC were cultured in vitro with hUCMSC or alone. The expression of IL-17 in CD4(+) T cells or γ/δ T cells were determined in each subject group by flow cytometry. IL-17 concentrations in PBMNC culture supernatants were measured by ELISA. The results indicated that the proportion of IL-17-producing CD4(+) T cells and IL-17-producing γ/δ T cells of SpA patients were 4.5 folds and 5 folds of healthy controls [CD3(+)CD4(+)IL-17(+) cells (3.42 ± 0.82)% vs (0.75 ± 0.25)%, P < 0.01; CD3(+)γδTCR(+)IL-17(+) cells (0.30 ± 0.10)% vs (0.06 ± 0.02)%, P < 0.01]. After co-culture of PBMNC in patients with hUCMSC, the increased proportions of CD3(+)CD4(+)IL-17(+) cells and CD3(+)γδTCR(+)IL-17(+) cells in SpA patients were inhibited significantly by hUCMSC [CD3(+)CD4(+)IL-17(+) cells (3.42 ± 0.82)% vs (1.81 ± 0.59)% (P < 0.01); CD3(+)γδTCR(+)IL-17(+) cells (0.30 ± 0.10)% vs (0.16 ± 0.06)% (P < 0.01]. In response to phytohemagglutinin (PHA, 1 µg/ml), PBMNC from SpA patients secreted more IL-17 than that from healthy control [(573.95 ± 171.68) pg/ml vs (115.53 ± 40.41) pg/ml (P < 0.01)]. In the presence of hUCMSC, PBMNC of SpA patients produced less amount of IL-17 [(573.95 ± 171.68) pg/ml vs (443.20 ± 147.94) pg/ml, (P < 0.01)]. It is concluded that the IL-17 production in peripheral blood T cells from SpA patients can be inhibited by hUCMSC, which have therapeutic potential for SpA.

Self-assembly of renal cells into engineered renal tissues in collagen/Matrigel scaffold in vitro.

To tissue engineer a kidney is a formidable task because of the complex cell composition and structures in the kidney. This study reconstructed renal tissues using mixed renal cells in collagen/Matrigel® scaffolds in vitro. Neonatal rat renal cells were seeded in collagen I supplemented with Matrigel in a casting mold that could exert static stretch when the renal constructs contracted. During in vitro culture, the renal constructs were observed under microscope and analyzed by histological and immunofluorescent examinations. Results showed that the mixed renal cells reconstituted renal tubular and glomeruli-like structures with different appearances at varying developmental stages. Tubular structures were formed by CK18-positive cells with similar appearances lining the surrounding hollow centres. The glomeruli-like structures were tufts of cell aggregates containing Flk-1-positive cells. These results show that neonatal rat renal cells self-assembled into engineered renal tissues containing both tubules and glomeruli-like structures when cultured in 3D collagen/Matrigel scaffold in vitro.

[Optimization of in vitro culture conditions for human amniotic epithelial cells and expression of stem cell markers].

This study was purposed to optimize the culture conditions of the human amniotic epithelium cells (hAEC) in vitro, and detect the expression of hAEC pluripotent markers. Amnion tissues were separated from the underlying chorion through the spongy layer immediately after elective cesarean section of healthy pregnancy women at term. After the subsequent exposure to trypsin digestion, hAEC were cultured in DMEM with different supplements. The growth and proliferation potential of hAEC was evaluated, and the expression of cultured hAEC pluripotent markers was detected by using flow cytometry and immunohistochemistry methods. The results indicated that when being cultured in the mediums similar to that of embryonic stem cell culture supplemented with 10 ng/ml EGF, the hAEC grew better and the time for passage was shortened. In addition, compared to other culture conditions, under this condition, the cells could be passaged up to 5 times as much without obvious morphological changes, and the pluripotent marker SSEA-4 was detected in the cultured cells by flow cytometry. Meanwhile, the detection of immunofluorescence showed the expression of vimentin in cultured hAEC was strengthened as compared with primary cells. It is concluded that the culture condition similar to that for embryonic stem cells supplemented with EGF facilitates the proliferation and passage of hAEC in vitro.

Reconstruction of endometrium in vitro via rabbit uterine endometrial cells expanded by sex steroid.

OBJECTIVE: To culture rabbit endometrial cells by using sex steroids to provide adequate seeding cells for endometrium reconstruction and uterine tissue engineering. DESIGN: Prospective experimental study. SETTING: Beijing Institute of Basic Medical Sciences and Tissue Engineering Research Center, Academy of Military Medical Sciences. ANIMAL(S): New Zealand rabbit and Kunming white strain mice. INTERVENTION(S): Rabbits were primed with pregnant mare serum gonadotropin and hCG. Endometrial cells were cultured with E(2) and P(4) of different concentrations. The endometrium was reconstructed by using endometrial cells as seeding cells and collagen-basement membrane matrix as scaffolds. MAIN OUTCOME MEASURE(S): Assay with 93-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide, immunofluorescence staining, flow cytometric analysis, hematoxylin and eosin and immunohistochemical staining, and developmental rate of embryos. RESULT(S): The expression patterns of estrogen receptor and P receptor of rabbit endometrium were different before and after treatment with pregnant mare serum gonadotropin-hCG. One hundred nanomolar E(2) with 10 nmol/L P(4) facilitated the proliferation of epithelial cells whereas 100 nmol/L P(4) facilitated that of stromal cells. The epithelial cells could be stable if cultured for seven or eight passages. Cells in the epithelial layer of the reconstructed endometrium were cytokeratin positive. Some showed columnar morphology akin to the luminal epithelium in vivo. Reconstructed endometrium could improve the developmental rate and quality of one-cell mice embryos. CONCLUSION(S): Rabbit endometrial cells could be cultured with a long-standing proliferation capability by sex steroids and applied in uterine tissue engineering. Reconstructed endometrium with proliferated endometrial cells was akin to native endometrium in structure and function.

Reconstruction of engineered uterine tissues containing smooth muscle layer in collagen/matrigel scaffold in vitro.

OBJECTIVE: This study attempted to reconstruct engineered uterine tissues (EUTs) containing smooth muscle layer, akin to the normal uterine wall. METHODS: EUTs were reconstructed by seeding epithelial cells on top of the constructed stromal layer over smooth muscle layer. A self-made mold was used to keep the EUTs from contraction. At the same time, it provided static stretch to the EUTs. After 14 days of culture, the structure of the EUTs was analyzed histologically and immunohistochemically, or by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The expression of integrin beta3 subunit, heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), and HOXA-10 was detected by reverse transcription-polymerase chain reaction (RT-PCR). The ability of the EUTs supporting the development of embryos was estimated by coculturing embryos on the EUTs. We also tried a new method to reconstruct EUTs by mixing epithelial cell and stromal cells (1:2) in collagen/Matrigel to form an endometrial layer and putting it on top of the smooth muscle layer. The self-assembling ability of the endometrial epithelial cells and stromal cells in the reconstructed EUTs was analyzed histologically and immunohistochemically. RESULTS: The results found that the constructed EUTs with the first and the second method showed three-layered structures. The epithelial layer, stromal layer, and smooth muscle layer were stained by cytokeratin 18, vimentin, and alpha-actin, respectively. TEM showed that the cells in the EUTs reconstructed by the first method were attached to each other by apical tight junctions and rivet-like desmosomes. SEM showed protruded pinopodes, microvilli, and cilium of epithelial cells. The RT-PCR analysis showed that integrin beta3 subunit, HB-EGF, and HOXA-10 were expressed in EUTs. The coculture system of EUTs improved the development rate and quality of murine embryo significantly in comparison with those of control Chatot Ziomek Bavister culture. In the EUTs reconstructed by the second method, the epithelial cells demonstrated self-assembling ability and formed epithelial cell layer on top of the stromal layer and glandular tube-like structures in the stromal layer. Columnar epithelial cells existed in some parts of the epithelial layer. CONCLUSION: We engineered EUTs containing smooth muscle layer by two methods. The reconstructed EUTs could support the development of embryos. The epithelial cells showed self-assembling ability in the EUTs.

Functional improvement of infarcted heart by co-injection of embryonic stem cells with temperature-responsive chitosan hydrogel.

Transplantation of embryonic stem cells (ESCs) can improve cardiac function in treatment of myocardial infarction. The low rate of cell retention and survival within the ischemic tissues makes the application of cell transplantation techniques difficult. In this study, we used a temperature-responsive chitosan hydrogel (as scaffold) combined with ESCs to maintain viable cells in the infarcted tissue. Temperature-responsive chitosan hydrogel was prepared and injected into the infarcted heart wall of rat infarction models alone or together with mouse ESCs. The result showed that the 24-h cell retention and 4 week graft size of both groups was significantly greater than with a phosphate buffered saline control. After 4 weeks of implantation, heart function, wall thickness, and microvessel densities within the infarct area improved in the chitosan + ESC, chitosan, and ESC group more than the PBS control. Of the three groups, the chitosan + ESC performed best. Results of this study indicate that temperature-responsive chitosan hydrogel is an injectable scaffold that can be used to deliver stem cells to infarcted myocardium. It can also increase cell retention and graft size. Cardiac function is well preserved, too.

An investigation on the division of neuronal PC12 cells induced by nerve growth factor.

Neuronal PC12 cells induced by nerve growth factor (NGF) have been considered to be postmitotic and lack the ability to divide. However, in this study, we not only detected DNA synthesis but also observed cell division in some morphologically differentiated neuronal PC12 cells bearing long neurites. More interestingly, in addition to the division of perikaryon, the neurites located on the division site of the cell membrane also divided into two parts and were allocated to the two daughter cells. These results demonstrate that the morphologically differentiated neuronal PC12 cells still retain the ability to divide. This is the first report that neuronal PC12 cells as well as their neurites can divide.

[Induced-division of neurons derived from neural stem cells].

In order to explore if mature neurons derived from neural stem cells have the potentiality to divide, we utilized the chemical digestion method to disperse the adult rat brain tissue into single cells, and culture them in serum-free medium. After being cultured for about eight days in vitro, the neural stem cells were induced to differentiate into neurons. The neurons were further induced to divide. Utilizing the method of serial photograph and NF-160 immunocytochemistry, the processes of division of some neurons were recorded. At the same time, PCNA+NF-160 (or Chat, GABA, GAD) double label were used to investigate if the dividing-neurons were mature ones. After the neural stem cells were induced to differentiate in vitro for eight days, they possessed the shape and character of mature neurons. The differentiated neuron had a big nucleus and one or two distinct nucleolus in the nuclear. Within the perikaryon,there were a large amount of dense and Nissl body-like structure. Several long processes emerged from various locations of the cell body. Then, EGF and bFGF were added into the medium to induce division. After two days of induced-division, neuron-like cells were observed to divide; moreover, the number of neuron-like cells in the region increased continually. Immunocytochemistry demonstrated these cells were NF-160-positive. Serial photographs of dividing-process of neuron-like cells were obtained and their daughter cells were also NF-160-positive. After PCNA+NF-160 (or Chat, GABA, GAD) double label, some cells showed brown cell plasma and black nucleus. The above-mentioned results indicate that neurons, which were previously thought to be end-differentiated, can be re-called into cell cycle under appropriate conditions. Mature neurons still have the potential to divide, proliferate and self-renew.

Peptidergic innervation of human esophageal and cardiac carcinoma.

AIM: To investigate the distribution of neuropeptide-immunoreactive nerve fibers in esophageal and cardiac carcinoma as well as their relationship with tumor cells so as to explore if there is nerve innervation in esophageal and cardiac carcinoma. METHODS: Esophageal and cardiac carcinoma specimens were collected from surgical operation. One part of them were fixed immediately with 4 % paraformaldehyde and then cut with a cryostat into 40-microm-thick sections to perform immunohistochemical analysis. Antibodies of ten kinds of neuropeptide including calcitonin gene-related peptide (CGRP), galanin (GAL), substance P (SP), etc. were used for immunostaining of nerve fibers. The other part of the tumor specimens were cut into little blocks (1 mm(3)) and co-cultured with chick embryo dorsal root ganglia (DRG) to investigate if the tumor blocks could induce the neurons of DRG to extend processes, so as to probe into the possible reasons for the nerve fibers growing into tumors. RESULTS: Substantial amounts of neuropeptide including GAL-, NPY-, SP-immunoreactive nerve bundles and scattered nerve fibers were distributed in esophageal and cardiac carcinomas. The scattered nerve fibers waved their way among tumor cells and contacted with tumor cells closely. Some of them even encircled tumor cells. There were many varicosities aligned on the nerve fibers like beads. They were also closely related to tumor cells. In the co-culture group, about 63 % and 67 % of DRG co-cultured with esophageal and cardiac tumor blocks respectively extended enormous processes, especially on the side adjacent to the tumor, whereas in the control group (without tumor blocks), no processes grew out. CONCLUSION: Esophageal and cardiac carcinomas may be innervated by peptidergic nerve fibers, and they can induce neurons of DRG to extend processes in vitro.