Stem Cells From Umbilical Cord Wharton's Jelly From Preterm Birth Have Neuroglial Differentiation Potential

Objective:The aim of the study is to determine the neuroglial differentiation potential of human Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) from preterm birth when compared to term delivery.Study Design:The WJ-MSCs from umbilical cords of preterm birth and term controls were isolated and induced into neural progenitors. The cells were analyzed for neuroglial markers by flow cytometry, real-time polymerase chain reaction, and immunocytochemistry. Results:Independent of gestational age, a subset of WJ-MSC displayed the neural progenitor cell markers Nestin and Musashi-1 and the mature neural markers microtubule-associated protein 2, glial fibrillary acidic protein, and myelin basic protein. Neuroglial induction of WJ-MSCs from term and preterm birth resulted in the enhanced transcription of Nestin and Musashi-1.Conclusions:Undifferentiated WJ-MSCs from preterm birth express neuroglial markers and can be successfully induced into neural progenitors similar to term controls. Their potential use as cellular graft in neuroregenerative therapy for peripartum brain injury in preterm birth has to be tested.

In vitro characterization of immune-related properties of human fetal bone cells for potential tissue engineering applications

We describe herein some immunological properties of human fetal bone cells recently tested for bone tissue-engineering applications. Adult mesenchymal stem cells (MSCs) and osteoblasts were included in the study for comparison. Surface markers involved in bone metabolism and immune recognition were analyzed using flow cytometry before and after differentiation or treatment with cytokines. Immunomodulatory properties were studied on activated peripheral blood mononuclear cells (PBMCs). The immuno-profile of fetal bone cells was further investigated at the gene expression level. Fetal bone cells and adult MSCs were positive for Stro-1, alkaline phosphatase, CD10, CD44, CD54, and beta2-microglobulin, but human leukocyte antigen (HLA)-I and CD80 were less present than on adult osteoblasts. All cells were negative for HLA-II. Treatment with recombinant human interferon gamma increased the presence of HLA-I in adult cells much more than in fetal cells. In the presence of activated PBMCs, fetal cells had antiproliferative effects, although with patterns not always comparable with those of adult MSCs and osteoblasts. Because of the immunological profile, and with their more-differentiated phenotype than of stem cells, fetal bone cells present an interesting potential for allogeneic cell source in tissue-engineering applications.

Transplantation of human embryonic stem cell-derived alveolar epithelial type II cells abrogates acute lung injury in mice.

Respiratory diseases are a major cause of mortality and morbidity worldwide. Current treatments offer no prospect of cure or disease reversal. Transplantation of pulmonary progenitor cells derived from human embryonic stem cells (hESCs) may provide a novel approach to regenerate endogenous lung cells destroyed by injury and disease. Here, we examine the therapeutic potential of alveolar type II epithelial cells derived from hESCs (hES-ATIICs) in a mouse model of acute lung injury. When transplanted into lungs of mice subjected to bleomycin (BLM)-induced acute lung injury, hES-ATIICs behaved as normal primary ATIICs, differentiating into cells expressing phenotypic markers of alveolar type I epithelial cells. Without experiencing tumorigenic side effects, lung injury was abrogated in mice transplanted with hES-ATIICs, demonstrated by recovery of body weight and arterial blood oxygen saturation, decreased collagen deposition, and increased survival. Therefore, transplantation of hES-ATIICs shows promise as an effective therapeutic to treat acute lung injury.

In vitro cell motility as a potential mesenchymal stem cell marker for multipotency.

Mesenchymal stem cells (MSCs) are expected to have a fundamental role in future cell-based therapies because of their high proliferative ability, multilineage potential, and immunomodulatory properties. Autologous transplantations have the "elephant in the room" problem of wide donor variability, reflected by variability in MSC quality and characteristics, leading to uncertain outcomes in the use of these cells. We propose life imaging as a tool to characterize populations of human MSCs. Bone marrow MSCs from various donors and in vitro passages were evaluated for their in vitro motility, and the distances were correlated to the adipogenic, chondrogenic, and osteogenic differentiation potentials and the levels of senescence and cell size. Using life-image measuring of track lengths of 70 cells per population for a period of 24 hours, we observed that slow-moving cells had the higher proportion of senescent cells compared with fast ones. Larger cells moved less than smaller ones, and spindle-shaped cells had an average speed. Both fast cells and slow cells were characterized by a low differentiation potential, and average-moving cells were more effective in undergoing all three lineage differentiations. Furthermore, heterogeneity in single cell motility within a population correlated with the average-moving cells, and fast- and slow-moving cells tended toward homogeneity (i.e., a monotonous moving pattern). In conclusion, in vitro cell motility might be a useful tool to quickly characterize and distinguish the MSC population's differentiation potential before additional use.

Derivation of pluripotent stem cells from cultured human primordial germ cells

Human pluripotent stem cells would be invaluable for in vitro studies of aspects of human embryogenesis. With the goal of establishing pluripotent stem cell lines, gonadal ridges and mesenteries containing primordial germ cells (PGCs, 5–9 weeks postfertilization) were cultured on mouse STO fibroblast feeder layers in the presence of human recombinant leukemia inhibitory factor, human recombinant basic fibroblast growth factor, and forskolin. Initially, single PGCs in culture were visualized by alkaline phosphatase activity staining. Over a period of 7–21 days, PGCs gave rise to large multicellular colonies resembling those of mouse pluripotent stem cells termed embryonic stem and embryonic germ (EG) cells. Throughout the culture period most cells within the colonies continued to be alkaline phosphatase-positive and tested positive against a panel of five immunological markers (SSEA-1, SSEA-3, SSEA-4, TRA-1–60, and TRA-1–81) that have been used routinely to characterize embryonic stem and EG cells. The cultured cells have been continuously passaged and found to be karyotypically normal and stable. Both XX and XY cell cultures have been obtained. Immunohistochemical analysis of embryoid bodies collected from these cultures revealed a wide variety of differentiated cell types, including derivatives of all three embryonic germ layers. Based on their origin and demonstrated properties, these human PGC-derived cultures meet the criteria for pluripotent stem cells and most closely resemble EG cells.

Stem cells in the periodontal ligament

The ability to identify and manipulate stem cells has been a significant advancement in regenerative medicine and has contributed to the development of tissue engineering-based clinical therapies. Difficulties associated with achieving predictable periodontal regeneration, means that novel techniques such as tissue engineering need to be developed in order to regenerate the extensive soft and hard tissue destruction that results from periodontitis. One of the critical requirements for a tissue engineering approach is the delivery of ex vivo expanded progenitor populations or the mobilization of endogenous progenitor cells capable of proliferating and differentiating into the required tissues. By definition, stem cells fulfill these requirements and the recent identification of stem cells within the periodontal ligament represents a significant development in the progress toward predictable periodontal regeneration. In order to explore the importance of stem cells in periodontal wound healing and regeneration, this review will examine contemporary concepts in stem cell biology, the role of periodontal ligament progenitor cells in the regenerative process, recent developments in identifying periodontal stem cells and the clinical implications of these findings.

A comparison of high-content screening versus manual analysis to assay the effects of mesenchymal stem cell-conditioned medium on neurite outgrowth in vitro

Bone marrow mesenchymal stem cells (MSCs) promote nerve growth and functional recovery in animal models of spinal cord injury (SCI) to varying levels. The authors have tested high-content screening to examine the effects of MSC-conditioned medium (MSC-CM) on neurite outgrowth from the human neuroblastoma cell line SH-SY5Y and from explants of chick dorsal root ganglia (DRG). These analyses were compared to previously published methods that involved hand-tracing individual neurites. Both methods demonstrated that MSC-CM promoted neurite outgrowth. Each showed the proportion of SH-SY5Y cells with neurites increased by ~200% in MSC-CM within 48 h, and the number of neurites/SH-SY5Y cells was significantly increased in MSC-CM compared with control medium. For high-content screening, the analysis was performed within minutes, testing multiple samples of MSC-CM and in each case measuring >15,000 SH-SY5Y cells. In contrast, the manual measurement of neurite outgrowth from >200 SH-SY5Y cells in a single sample of MSC-CM took at least 1 h. High-content analysis provided additional measures of increased neurite branching in MSC-CM compared with control medium. MSC-CM was also found to stimulate neurite outgrowth in DRG explants using either method. The application of the high-content analysis was less well optimized for measuring neurite outgrowth from DRG explants than from SH-SY5Y cells.

Establishment of a protocol for the cryopreservation of cell sheets of adipose tissue stem cells

Dissertação de Mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016

Mesenchymal Stromal Cells From Adipose Tissue Attached To Suture Material Enhance The Closure Of Enterocutaneous Fistulas In A Rat Model.

Surgical treatment for enterocutaneous fistulas (EF) frequently fails. Cell therapy may represent a new approach to treatment. Mesenchymal stromal cells (MSCs) have high proliferative and differentiation capacity. This study aimed to investigate whether MSCs could adhere to suture filament (SF), promoting better EF healing. MSCs, 1 × 10(6), from adipose tissue (ATMSCs) were adhered to a Polyvicryl SF by adding a specific fibrin glue formulation. Adhesion was confirmed by confocal and scanning electron microscopy (SEM). A cecal fistula was created in 22 Wistar rats by incising the cecum and suturing the opening to the surgical wound subcutaneously with four separate stitches. The animals were randomly allocated to three groups: control (CG)-five animals, EF performed; injection (IG)-eight animals 1 × 10(6) ATMSCs injected around EF borders; and suture filament (SG): nine animals, sutured with 1 × 10(6) ATMSCs attached to the filaments with fibrin glue. Fistulas were photographed on the operation day and every 3 days until the 21st day and analyzed by two observers using ImageJ Software. Confocal and SEM results demonstrated ATMSCs adhered to SF (ATMSCs-SF). The average reduction size of the fistula area at 21st day was greater for the SG group (90.34%, P < 0.05) than the IG (71.80%) and CG (46.54%) groups. ATMSCs adhered to SF maintain viability and proliferative capacity. EF submitted to ATMSCs-SF procedure showed greater recovery and healing. This approach might be a new and effective tool for EF treatment.

Protocols for obtainment and isolation of two mesenchymal stem cell sources in sheep

PURPOSE: To evaluate different protocols to isolate stem cells from ovine umbilical cord blood and adipose tissue. METHODS: There were used 5 samples of umbilical blood and 5 samples of perirenal adipose tissue from 10 female sheep. All the samples were obtained through surgery, to harvest aseptic samples. There were used 3 protocols for obtainment and culture of umbilical cord blood stem cells and 4 protocols for ovine adipose tissue stem cells. RESULTS: It was possible to observe only one successful protocol for the obtainment of umbilical cord blood stem cells. When analyzing the techniques used to obtain adipose tissue stem cells, only one of the methods was effective as well. Through colony forming unit assay, there were obtained 58 colonies of cells after seven days in culture. Flow citometry tests revealed the cells were positive to CD44 and exhibited negative reaction to CD38, CD45, CD41/61. These cells showed a growth curve with very well defined phases LOG, LAG and PLATEAU. This phases are typically seem in mesenchymal stem cells growth curves. CONCLUSIONS: The isolation and culture of mesenchymal stem cells from ovine umbilical cord blood are complex and request more detailed assays. Stem cells from fat tissue sheep showed mesenchymal characteristics, according to their cell growth curve, ability to origin colonies of fibroblastoid cells and positive reactivity with the antibody CD44 by flow citometry.

Mesenchymal Stem Cell Therapy in Heart Disease

Cardiovascular disease is among the main causes of mortality and morbidity worldwide. Despite significant advances in medical and interventional therapy, the prognosis of conditions such as ischemic heart disease is still dismal. There is thus a need to investigate new therapeutic tools, one of which is stem cell therapy. Hematopoietic stem cells are the most studied type, and the fact that their biology is relatively well understood has led to their being used in preclinical research and clinical trials. However, the results of some of these studies have been controversial, which has opened the way for studies on other cell types, such as mesenchymal stem cells. These cells have immunomodulatory properties which suggest that they have therapeutic potential in cardiology. In the present article, the authors review the state of the art regarding mesenchymal stem cells, from basic and translational research to their use in clinical trials on ischemic heart disease, heart failure and arrhythmias, and discuss possible future uses.

Overexpressing BDNF in Neural Stem Cells using non-viral gene delivery strategies

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Process Engineering of Stem Cells for Clinical Application

Over the last decade, human embryonic stem cells (hESCs) have garnered a lot of attention owing to their inherent self-renewal ability and pluripotency. These characteristics have opened opportunities for potential stem cell-based regenerative medicines, for development of drug discovery platforms and as unique in vitro models for the study of early human development.(...)

Development of Janus hydrogel microcapsules as novel biomaterials-stem cells construct for 3D co-culture applications

Co-cultures of two or more cell types and biodegradable biomaterials of natural origin have been successfully combined to recreate tissue microenvironments. Segregated co-cultures are preferred over conventional mixed ones in order to better control the degree of homotypic and heterotypic interactions. Hydrogel-based systems in particular, have gained much attention to mimic tissue-specific microenvironments and they can be microengineered by innovative bottom-up approaches such as microfluidics. In this study, we developed bi-compartmentalized (Janus) hydrogel microcapsules of methacrylated hyaluronic acid (MeHA)/methacrylated-chitosan (MeCht) blended with marine-origin collagen by droplet-based microfluidics co-flow. Human adipose stem cells (hASCs) and microvascular endothelial cells (hMVECs) were co-encapsulated to create platforms of study relevant for vascularized bone tissue engineering. A specially designed Janus-droplet generator chip was used to fabricate the microcapsules (<250â μm units) and Janus-gradient co-cultures of hASCs: hMVECs were generated in various ratios (90:10; 75:25; 50:50; 25:75; 10:90), through an automated microfluidic flow controller (Elveflow microfluidics system). Such monodisperse 3D co-culture systems were optimized regarding cell number and culture media specific for concomitant maintenance of both phenotypes to establish effective cell-cell (homotypic and heterotypic) and cell-materials interactions. Cellular parameters such as viability, matrix deposition, mineralization and hMVECs re-organization in tube-like structures, were enhanced by blending MeHA/MeCht with marine-origin collagen and increasing hASCs: hMVECs co-culture gradient had significant impact on it. Such Janus hybrid hydrogel microcapsules can be used as a platform to investigate biomaterials interactions with distinct combined cell populations.

Chondrogenic potential of injectable κ-carrageenan hydrogel with encapsulated adipose stem cells for cartilage tissue-engineering applications

Due to the limited self-repair capacity of cartilage, regenerative medicine therapies for the treatment of cartilage defects must use a significant amount of cells, preferably applied using a hydrogel system that can promise their delivery and functionality at the specific site. This paper discusses the potential use of k-carrageenan hydrogels for the delivery of stem cells obt ained from adipose tissue in the treatment of cartilage tissue defects. The developed hydrogels were produced by an ionotropic gelation met hod and human adipose stem cells (hASCs) were encapsulated in 1.5% w/v k-carrageenan solution at a cell density of 5  10 6 cells/ml. The results from the analysis of the cell-encapsulating hydrogels, cultured for up to 21 days, indicated that k-carrageenan hydrogels support the viability, proliferation and chondrogenic differentiation of hASCs. Additionally, the mec hanical analysis demonstrated an increase in stiffness and viscoelastic properties of k-carrageenan gels with their encapsulated cells with increasing time in culture with chondrogenic medium. These results allowed the conclusion that k-carrageenan exhibits properties t hat enable the in vitro functionality of encapsulated hASCs and thus may provide the basis for new successful approaches for the treatment of cartilage defects.