Wharton's jelly-derived mesenchymal cells as a new source for the generation of microtissues for tissue engineering applications.

Microtissues (MT) are currently considered as a promising alternative for the fabrication of natural, 3D biomimetic functional units for the construction of bio-artificial substitutes by tissue engineering (TE). The aim of this study was to evaluate the possibility of generating mesenchymal cell-based MT using human umbilical cord Wharton's jelly stromal cells (WJSC-MT). MT were generated using agarose microchips and evaluated ex vivo during 28 days. Fibroblasts MT (FIB-MT) were used as control. Morphometry, cell viability and metabolism, MT-formation process and ECM synthesis were assessed by phase-contrast microscopy, functional biochemical assays, and histological analyses. Morphometry revealed a time-course compaction process in both MT, but WJSC-MT resulted to be larger than FIB-MT in all days analyzed. Cell viability and functionality evaluation demonstrated that both MT were composed by viable and metabolically active cells, especially the WJSC during 4-21 days ex vivo. Histology showed that WJSC acquired a peripheral pattern and synthesized an extracellular matrix-rich core over the time, what differed from the homogeneous pattern observed in FIB-MT. This study demonstrates the possibility of using WJSC to create MT containing viable and functional cells and abundant extracellular matrix. We hypothesize that WJSC-MT could be a promising alternative in TE protocols. However, future cell differentiation and in vivo studies are still needed to demonstrate the potential usefulness of WJSC-MT in regenerative medicine.

Membranes derived from human umbilical cord Wharton's jelly stem cells as novel bioengineered tissue-like constructs.

Cell-derived matrices were recently described as novel biomaterials generated by human cells allowed to grow and synthetize their own extracellular matrix in culture. In the present work, we generated and evaluated a novel tissue-like substitute (WDM) consisting of a membrane derived from cultured human Wharton's jelly stem cells. WDM were evaluated ex vivo and in vivo by histochemistry and immunohistochemistry for several mesenchymal cell markers and fibrillar and non-fibrillar extracellular matrix components. Results show that WDM were heterogeneous and consisted of dense cell-poor areas surrounded by cell-rich zones with abundant HWJSC. Histological analyses demonstrated that cell-poor areas were very rich in fibrillar and non-fibrillar extracellular matrix components such as collagen and proteoglycans, and cells in the WDM were highly viable and mostly PCNA-positive. HWJSC in the WDM expressed all markers of this cell type, including CD90, CD105, pan cytokeratin and CK8. In vivo analysis showed that the WDM was highly biocompatible and grafting this membrane in the muscle of laboratory rats was not associated to increased inflammation, necrosis, tumorigenesis or other side effects, while cells properly integrated at the damage site and showed high proliferation index. These results suggest that the structure and composition of the extracellular matrix of these novel WDM could reproduce the situation of native human tissues and that WDM implanted in vivo are highly biocompatible and rapidly integrate in the host tissues. For these reasons, we hypothesize that WDM could be used in regenerative medicine protocols.

Bioactive injectable aggregates with nanofibrous microspheres and human dental pulp stem cells: A translational strategy in dental endodontics.

Regeneration of the pulp-dentin complex with stem cells is a potential alternative to conventional root canal treatments. Human dental pulp stem cells (hDPSCs) have been extensively studied because of their ability to proliferate and differentiate into mineralized dental and non-dental tissues. Here we combined hDPSCs with two types of injectable poly-l-lactic acid (PLLA) microsphere with a nanofibrous or smooth surface to form bioactive injectable aggregates, and examined their ability to promote pulp regeneration in the root canal in an in vivo model. We investigated the biocompatibility, biosafety and odontogenic potential of fibrous (F-BIM) and smooth bioactive injectable microspheres (S-BIM) in vitro and in vivo. Our results demonstrated that PLLA microspheres and hDPSCs were able to form bioactive injectable aggregates that promoted dentin regeneration in both in vitro and in vivo models. Our results suggest that F-BIM and S-BIM may induce dentinogenesis upon in vivo grafting, and propose that the potential usefulness of the microsphere-hDPSC aggregates described here should be evaluated in clinical settings. Copyright © 2017 John Wiley & Sons, Ltd.

Expression of epithelial markers by human umbilical cord stem cells. A topographical analysis.

INTRODUCTION: Human umbilical cord stem cells have inherent differentiation capabilities and potential usefulness in regenerative medicine. However, the epithelial differentiation capability and the heterogeneity of these cells have not been fully explored to the date. METHODS: We analyzed the expression of several undifferentiation and epithelial markers in cells located in situ in different zones of the umbilical cord -in situ analysis- and in primary ex vivo cell cultures of Wharton's jelly stem cells by microarray and immunofluorescence. RESULTS: Our results demonstrated that umbilical cord cells were heterogeneous and had intrinsic capability to express in situ stem cell markers, CD90 and CD105 and the epithelial markers cytokeratins 3, 4, 7, 8, 12, 13, 19, desmoplakin and zonula occludens 1 as determined by microarray and immunofluorescence, and most of these markers remained expressed after transferring the cells from the in situ to the ex vivo cell culture conditions. However, important differences were detected among some cell types in the umbilical cord, with subvascular zone cells showing less expression of stem cell markers and cells in Wharton's jelly and the amnioblastic zones showing the highest expression of stem cells and epithelial markers. CONCLUSIONS: These results suggest that umbilical cord mesenchymal cells have intrinsic potential to express relevant epithelial markers, and support the idea that they could be used as alternative cell sources for epithelial tissue engineering.

Aislamiento, cultivo y aplicación clínica de los melanocitos humanos: Los melanocitos como una fuente de terapia celular / Isolation, culture and clinical application of human melanocytes: Melanocytes as a source of cell therapy

El vitiligo es una enfermedad pigmentaria cutánea adquirida e idiopática, debido a múltiples factores causales que ocasionan la destrucción o inactivación funcional del melanocito. Ha habido numerosas alternativas terapéuticas a lo largo de los años como: tratamientos tópicos, PUVA (psoralenos más luz ultravioleta) o fototerapia UVB de banda estrecha; sin embargo, muchos no responden a estos tratamientos médicos. El tratamiento quirúrgico del vitiligo se indica cuando las máculas hipo o amelanóticas, se localizan en zonas mal respondedoras o tras fracaso de los tratamientos convencionales. Se trata de favorecer la re-pigmentación de las lesiones, a través del trasplante de melanocitos desde las áreas pigmentadas normales. El objetivo de esta revisión, es el de analizar el estado actual del tratamiento quirúrgico del vitiligo y en concreto, del trasplante de melanocitos autólogos.

Vitiligo is an idiopathic and acquired disorder that affects the pigmentation of the skin due to several factors that cause the destruction of melanocytes. There are numerous therapeutic alternatives, topical treatments, PUVA or narrowband UVB phototherapy. However, many patients do not respond to these medical treatments. Surgical treatment of vitiligo is indicated when depigmented macules are located in poorly responsive or after failure of conventional treatments. It seeks to encourage repigmentation of lesions by transplantation of melanocytes from normal pigmented areas. The objective of this review is to analyze surgical treatment of vitiligo and especially autologous melanocyte transplantation.

El folículo piloso: una importante fuente celular en ingeniería tisular / Hair follicle: an important cellular source in tissue engineering

El folículo piloso-sebáceo o unidad folículo-sebácea-apócrina es un órgano microscópico complejo funcional y estructuralmente. Consta de un segmento superior, estable y un segmento inferior, que se regenera en cada ciclo piloso. El folículo piloso contiene una gran diversidad de tipos celulares, entre los que se encuentran células madre. De hecho, supone el nicho más importante de células madre en la piel, con la ventaja añadida de su fácil accesibilidad. Estas células madre se localizan en una prominencia a nivel de la inserción del músculo erector del pelo, llamada "bulge" (promontorio). Son las encargadas de regenerar el folículo en cada ciclo y también intervienen en la reconstitución de las glándulas sebáceas y de la epidermis interfolicular, en caso de lesión epidérmica. El conocimiento de la estructura del folículo piloso, está haciendo posible cada vez más el empleo de los distintos tipos celulares, especialmente, las células madre, en la ingeniería tisular de la piel. Asimismo, estudios recientes han diferenciado las células madres foliculares a diferentes estirpes, como por ejemplo: células nerviosas, hematopoyéticas y vasculares. Además, se ha estudiado la construcción de folículos pilosos, con resultados satisfactorios en ratones, aunque no del todo superponibles a humanos, en los que se precisan más investigaciones. La posibilidad de generar folículos pilosos humanos, supondría una revolución en el amplio campo de las alopecias. El objetivo de esta revisión es describir la estructura anatómica e histológica del folículo piloso, enfatizando en la importancia del mismo como nicho de células madre y su potencial utilidad en el campo de la ingeniería tisular, para la construcción de diversos tipos de tejidos.

The hair follicle is a microscopic organ, functionally and structurally complex. It can be divided into two distinct segments, the upper portion, stable and the lower portion, that undergoes regeneration every hair cycle. The hair follicle contains a variety of cells types, including stem cells. It fact, it is the most important niche of stem cells in the skin, with the added advantage of its easy accessibility. These stem cells are located in a prominence at the level of the insertion of the arrector pili muscle, called bulge. They are responsible of hair follicle regeneration each hair cycle, and also can form sebaceous glands and help in repopulation of the interfollicular epidermis after injury. The knowledge of hair follicle structure is making possible the use of different types of cells, especially stem cells, in skin tissue engineering. Furthermore, recent studies have differentiated follicle stem cells into different strains, for example: nervous, hematopoietic and vascular cells. Moreover, other late studies have focused on the hair follicle construction, with satisfactory results in mice, but not completely transferable to humans, which further research is needed. The possibility of human hair follicle regeneration would suppose a revolution in the broad world of alopecia. The aim of this review is to describe the anatomical and histological structure of the hair follicle, emphasizing the importance as stem cell niche, and its potential usefulness in the field of tissue engineering for the construction of various types of tissues.

MT1 melatonin receptor expression in Warthin's tumor.

We contribute the first immunohistochemical study of MT1 melatonin receptor in Warthin's tumor and normal parotid gland. All 14 Warthin's tumors studied showed intense cytoplasmic positivity for MT1 receptor in all cylindrical epithelial cells lining spaces and a less intense positivity in basal cells. The lymphoid component accompanying the tumor was always negative for MT1 receptor. The parotid structure surrounding the tumor showed intense cytoplasmic positivity in all cells lining excretory ducts (lobar and lobulillar), with a lesser and focal positivity in cells of the acinar component. The biological activity of MT1 receptor in epithelial cells lining parotid excretory ducts may resemble its activity in Warthin's tumor cells. Hence, we propose Warthin's tumor as a useful positive control in immunohistochemical studies of MT1 melatonin receptor.

Expression of smoothelin and smooth muscle actin in the skin.

INTRODUCTION: Smoothelin is a cytoskeletal protein of differentiated smooth muscle cells with contractile capacity, distinguishing it from other smooth muscle proteins, such as smooth muscle actin (SMA). OBJECTIVE: To evaluate the expression of smoothelin and SMA in the skin in order to establish specific localizations of smoothelin in smooth muscle cells with high contractile capacity and in the epithelial component of cutaneous adnexal structures. METHODS: Immunohistochemical analysis (smoothelin and SMA) was performed in 18 patients with normal skin. RESULTS: SMA was expressed by the vascular structures of superficial, deep, intermediate and adventitial plexuses, whereas smoothelin was specifically expressed in the cytoplasm of smooth muscle cells of the deepest vascular plexus and in no other plexus of the dermis. The hair erector muscle showed intense expression of smoothelin and SMA. Cells with nuclear expression of smoothelin and cytoplasmic expression of SMA were observed in the outer root sheath of the inferior portion of the hair follicles and intense cytoplasmic expression in cells of the dermal sheath to SMA. CONCLUSIONS: We report the first study of smoothelin expression in normal skin, which differentiates the superficial vascular plexus from the deep. The deep plexus comprises vessels with high contractile capacity, which is important for understanding dermal hemodynamics in normal skin and pathological processes. We suggest that the function of smoothelin in the outer root sheath may be to enhance the function of SMA, which has been related to mechanical stress. Smoothelin has not been studied in cutaneous pathology; however we believe it may be a marker specific for the diagnosis of leiomyomas and leiomyosarcomas of the skin. Also, smoothelin could differentiate arteriovenous malformations of cavernous hemangioma of the skin.

Maternal diabetes affects specific extracellular matrix components during placentation.

During embryo implantation, invasive trophoblast cells mediate embryo invasion into the decidualized stroma, forming a rich network of lacunae that connect the embryonic tissues to the maternal blood vessels. Placentation is probably guided by the composition and organization of the endometrial extracellular matrix. Certain pathological conditions that occur during pregnancy, including diabetes, have been linked to abnormal placental morphology and consequent fetal morbidity. We used immunoperoxidase techniques to identify members of the collagen, proteoglycan and glycoprotein families in the various compartments of the rat placenta and to determine whether experimentally induced diabetes affects placental morphology and alters the distribution of these molecules during pregnancy. Single injections of alloxan (40 mg kg(-1) i.v.) were used to induce diabetes on day 2 of pregnancy in Wistar rats. Placentas were collected on days 14, 17, and 20. Type I and III collagen, as well as the proteoglycans decorin and biglycan, were found to be distributed throughout the placentas of control and diabetic rats. In both groups, laminin expression decreased at the end of pregnancy. In contrast, fibronectin was detected in the labyrinth region of diabetic rats at all gestational stages studied, whereas it was detected only at term pregnancy in the placentas of control rats. These results show for the first time that some extracellular matrix molecules are modulated during placental development. However, as diabetic rats presented increased fibronectin deposition exclusively in the labyrinth region, we speculate that diabetes alters the microenvironment at the maternal-fetal interface, leading to developmental abnormalities in the offspring.