Vascular Progenitor Cells Isolated from Human Embryonic Stem Cells

Invariant left-right (LR) patterning or chirality is critical for embryonic development. The loss or reversal of LR asymmetry is often associated with malformations and disease. Although several theories have been proposed, the exact mechanism of the initiation of the LR symmetry has not yet been fully elucidated. Recently, chirality has been detected within single cells as well as multicellular structures using several in vitro approaches. These studies demonstrated the universality of cell chirality, its dependence on cell phenotype, and the role of physical boundaries. In this review, we discuss the theories for developmental LR asymmetry, compare various in vitro cell chirality model systems, and highlight possible roles of cell chirality in stem cell differentiation. We emphasize that the in vitro cell chirality systems have great promise for helping unveil the nature of chiral morphogenesis in development

Engineering of human cardiac muscle electromechanically matured to an adult-like phenotype

Author ManuscriptThe application of tissue-engineering approaches to human induced pluripotent stem (hiPS) cells enables the development of physiologically relevant human tissue models for in vitro studies of development, regeneration, and disease. However, the immature phenotype of hiPS-derived cardiomyocytes (hiPS-CMs) limits their utility. We have developed a protocol to generate engineered cardiac tissues from hiPS cells and electromechanically mature them toward an adult-like phenotype. This protocol also provides optimized methods for analyzing these tissues' functionality, ultrastructure, and cellular properties. The approach relies on biological adaptation of cultured tissues subjected to biomimetic cues, applied at an increasing intensity, to drive accelerated maturation. hiPS cells are differentiated into cardiomyocytes and used immediately after the first contractions are observed, when they still have developmental plasticity. This starting cell population is combined with human dermal fibroblasts, encapsulated in a fibrin hydrogel and allowed to compact under passive tension in a custom-designed bioreactor. After 7 d of tissue formation, the engineered tissues are matured for an additional 21 d by increasingly intense electromechanical stimulation. Tissue properties can be evaluated by measuring contractile function, responsiveness to electrical stimuli, ultrastructure properties (sarcomere length, mitochondrial density, networks of transverse tubules), force-frequency and force-length relationships, calcium handling, and responses to β-adrenergic agonists. Cell properties can be evaluated by monitoring gene/protein expression, oxidative metabolism, and electrophysiology. The protocol takes 4 weeks and requires experience in advanced cell culture and machining methods for bioreactor fabrication. We anticipate that this protocol will improve modeling of cardiac diseases and testing of drugs.NIBIB and NCATS grant EB17103 (G.V.-N.); NIBIB, NCATS, NIAMS, NIDCR, and NIEHS grant EB025765 (G.V.-N.); NHLBI grants HL076485 (G.V.-N.) and HL138486 (M.Y.); NSF grant 16478 (G.V.-N.); the University of Minho MD/PhD program (D.T.); a Japan Society for the Promotion of Science fellowship (K.M.); and the Columbia University Stem Cell Initiative (L.S., M.Y.

Percutaneous Cell Delivery Into the Heart Using Hydrogels Polymerizing In Situ

Heart disease is the leading cause of death in the US. Following an acute myocardial infarction, a fibrous, noncontractile scar develops, and results in congestive heart failure in more than 500,000 patients in the US each year. Muscle regeneration and the induction of new vascular growth to treat ischemic disorders of the heart can have significant therapeutic implications. Early studies in patients with chronic ischemic systolic left ventricular dysfunction (SLVD) using skeletal myoblasts or bone marrow-derived cells report improvement in left ventricular ejection function (LVEF) and clinical status, without notable safety issues. Nonetheless, the efficacy of cell transfer for cardiovascular disease is not established, in part due to a lack of control over cell retention, survival, and function following delivery. We studied the use of biocompatible hydrogels polymerizable in situ as a cell delivery vehicle, to improve cell retention, survival, and function following delivery into the ischemic myocardium. The study was conducted using human bone marrow-derived mesenchymal stem cells and fibrin glue, but the methods are applicable to any human stem cells (adult or embryonic) and a wide range of hydrogels. We first evaluated the utility of several commercially available percutaneous catheters for delivery of viscous cell/hydrogel suspensions. Next we characterized the polymerization kinetics of fibrin glue solutions to define the ranges of concentrations compatible with catheter delivery. We then demonstrate the in vivo effectiveness of this preparation and its ability to increase cell retention and survival in a nude rat model of myocardial infarction

Geometric Control of Human Stem Cell Morphology and Differentiation

During tissue morphogenesis, stem cells and progenitor cells migrate, proliferate, and differentiate, with striking changes in cell shape, size, and acting mechanical stresses. The local cellular function depends on the spatial distribution of cytokines as well as local mechanical microenvironments in which the cells reside. In this study, we controlled the organization of human adipose derived stem cells using micro-patterning technologies, to investigate the influence of multi-cellular form on spatial distribution of cellular function at an early stage of cell differentiation. The underlying role of cytoskeletal tension was probed through drug treatment. Our results show that the cultivation of stem cells on geometric patterns resulted in pattern- and position-specific cell morphology, proliferation and differentiation. The highest cell proliferation occurred in the regions with large, spreading cells (such as the outer edge of a ring and the short edges of rectangles). In contrast, stem cell differentiation co-localized with the regions containing small, elongated cells (such as the inner edge of a ring and the regions next to the short edges of rectangles). The application of drugs that inhibit the formation of actomyosin resulted in the lack of geometrically specific differentiation patterns. This study confirms the role of substrate geometry on stem cell differentiation, through associated physical forces, and provides a simple and controllable system for studying biophysical regulation of cell function

Development of silk-based scaffolds for tissue engineering of bone from human adipose derived stem cells

Silk fibroin is a potent alternative to other biodegradable biopolymers for bone tissue engineering (TE), because of its tunable architecture and mechanical properties, and its demonstrated ability to support bone formation both in vitro and in vivo. In this study, we investigated a range of silk scaffolds for bone TE using human adipose-derived stem cells (hASCs), an attractive cell source for engineering autologous bone grafts. Our goal was to understand the effects of scaffold architecture and biomechanics and use this information to optimize silk scaffolds for bone TE applications. Silk scaffolds were fabricated using differ- ent solvents (aqueous vs. hexafluoro-2-propanol (HFIP)), pore sizes (250–500 um vs. 500–1000 um) and structures (lamellar vs. spherical pores). Four types of silk scaffolds combining the properties of interest were systematically compared with respect to bone tissue outcomes, with decellularized trabecular bone (DCB) included as a ‘‘gold standard’’. The scaffolds were seeded with hASCs and cultured for 7 weeks in osteogenic medium. Bone formation was evaluated by cell proliferation and differentiation, matrix production, calcification and mechanical properties. We observed that 400–600 um porous HFIP-derived silk fibroin scaffold demonstrated the best bone tissue formation outcomes, as evidenced by increased bone protein production (osteopontin, collagen type I, bone sialoprotein), enhanced calcium deposition and total bone volume. On a direct comparison basis, alkaline phosphatase activity (AP) at week 2 and new calcium deposition at week 7 were comparable to the cells cultured in DCB. Yet, among the aqueous- based structures, the lamellar architecture induced increased AP activity and demonstrated higher equi- librium modulus than the spherical-pore scaffolds. Based on the collected data, we propose a conceptual model describing the effects of silk scaffold design on bone tissue formation.FCT: SFRH/BD/42316/2007NIH: DE161525 and EB0252

Advanced maturation of human cardiac tissue grown from pluripotent stem cells

Cardiac tissues generated from human induced pluripotent stem cells (iPSCs) can serve as platforms for patient-specific studies of physiology and disease1-6. However, the predictive power of these models is presently limited by the immature state of the cells1, 2, 5, 6. Here we show that this fundamental limitation can be overcome if cardiac tissues are formed from early-stage iPSC-derived cardiomyocytes soon after the initiation of spontaneous contractions and are subjected to physical conditioning with increasing intensity over time. After only four weeks of culture, for all iPSC lines studied, such tissues displayed adult-like gene expression profiles, remarkably organized ultrastructure, physiological sarcomere length (2.2 µm) and density of mitochondria (30%), the presence of transverse tubules, oxidative metabolism, a positive force-frequency relationship and functional calcium handling. Electromechanical properties developed more slowly and did not achieve the stage of maturity seen in adult human myocardium. Tissue maturity was necessary for achieving physiological responses to isoproterenol and recapitulating pathological hypertrophy, supporting the utility of this tissue model for studies of cardiac development and disease.The authors acknowledge funding support from the National Institutes of Health of the USA (NIBIB and NCATS grant EB17103 (G.V.-N.); NIBIB, NCATS, NIAMS, NIDCR and NIEHS grant EB025765 (G.V.-N.); NHLBI grants HL076485 (G.V.-N.) and HL138486 (M.Y.); Columbia University MD/PhD program (S.P.M., T.C.); University of Minho MD/PhD program (D.T.); Japan Society for the Promotion of Science fellowship (K.M.); and Columbia University Stem Cell Initiative (D.S., L.S., M.Y.). We thank S. Duncan and B. Conklin for providing human iPSCs, M.B. Bouchard for assistance with image and video analysis, and L. Cohen-Gould for transmission electron microscopy services.info:eu-repo/semantics/publishedVersio

Stem cells from human extracted deciduous teeth expanded in foetal bovine and human sera express different paracrine factors after exposure to freshly prepared human serum

Background: The response of stem cells to paracrine factors within the host’s body plays an important role in the regeneration process after transplantation. The aim of this study was to determine the viability and paracrine factor profile of stem cells from human extracted deciduous teeth (SHED) pre-cultivated in media supplemented with either foetal bovine serum (FBS) or pooled human serum (pHS) in the presence of individual human sera (iHS). Methods: SHED (n=3) from passage 4 were expanded in FBS (FBS-SHED) or pHS (pHS-SHED) supplemented media until passage 7. During expansion, the proliferation of SHED was determined. Cells at passage 7 were further expanded in human serum from four individual donors (iHS) for 120 hours followed by assessment of cell viability and profiling of the secreted paracrine factors. Results: Proliferation of SHED was significantly higher (p<0.05) in pHS supplemented media compared to FBS supplemented media. pHS-SHED also maintained their higher proliferation rate compared to FBS-SHED in the presence of iHS. In iHS supplemented media, FBS-SHED expressed significantly higher levels of SDF-1A (p<0.05) after 24 hours compared to pHS-SHED. Similar results were found for HGF (p<0.01), LIF (p<0.05), PDGF-BB (p<0.05), SDF-1A (p<0.01), and IL-10 (p<0.05) when cell culture supernatants from FBS-SHED was profiled 120 hours post-incubation. Conclusion: SHED expanded in pHS instead of FBS have higher proliferative capacity and show an altered secretion profile. Further studies are needed to determine whether these differences could result in better engraftment and regeneration following transplantation

Bioreactors as engineering support to treat cardiac muscle and vascular disease

Cardiovascular disease is the leading cause of morbidity and mortality in the Western World. The inability of fully differentiated, load-bearing cardiovascular tissues to in vivo regenerate and the limitations of the current treatment therapies greatly motivate the efforts of cardiovascular tissue engineering to become an effective clinical strategy for injured heart and vessels. For the effective production of organized and functional cardiovascular engineered constructs in vitro, a suitable dynamic environment is essential, and can be achieved and maintained within bioreactors. Bioreactors are technological devices that, while monitoring and controlling the culture environment and stimulating the construct, attempt to mimic the physiological milieu. In this study, a review of the current state of the art of bioreactor solutions for cardiovascular tissue engineering is presented, with emphasis on bioreactors and biophysical stimuli adopted for investigating the mechanisms influencing cardiovascular tissue development, and for eventually generating suitable cardiovascular tissue replacements