Tabassum Ahsan

Shear stress during early embryonic stem cell differentiation promotes hematopoietic and endothelial phenotypes

Pluripotent embryonic stem cells (ESCs) are a potential source for cell‐based tissue engineering and regenerative medicine applications, but their translation into clinical use will require efficient and robust methods for promoting differentiation. Fluid shear stress, which can be readily incorporated into scalable bioreactors, may be one solution for promoting endothelial and hematopoietic phenotypes from ESCs. Here we applied laminar shear stress to differentiating ESCs using a 2D adherent parallel plate configuration to systematically investigate the effects of several mechanical parameters. Treatment similarly promoted endothelial and hematopoietic differentiation for shear stress magnitudes ranging from 1.5 to 15 dyne/cm2 and for cells seeded on collagen‐, fibronectin‐ or laminin‐coated surfaces. Extension of the treatment duration consistently induced an endothelial response, but application at later stages of differentiation was less effective at promoting hematopoietic phenotypes. Furthermore, inhibition of the FLK1 protein (a VEGF receptor) neutralized the effects of shear stress, implicating the membrane protein as a critical mediator of both endothelial and hematopoietic differentiation by applied shear. Using a systematic approach, studies such as these help elucidate the mechanisms involved in force‐mediated stem cell differentiation and inform scalable bioprocesses for cellular therapies. Biotechnol. Bioeng. 2013; 110: 1231–1242.

An angiogenesis model for investigating multicellular interactions across intact microvascular networks

Developing therapies aimed at manipulating microvascular remodeling requires a better understanding of angiogenesis and how angiogenesis relates to other network remodeling processes, such as lymphangiogenesis and neurogenesis. The objective of this study was to develop an angiogenesis model that enables probing of multicellular and multisystem interactions at the molecular level across an intact adult microvascular network. Adult male Wistar rat mesenteric windows were aseptically harvested and cultured in serum-free minimum essential media. Viability/cytotoxicity analysis revealed that cells remain alive for at least 7 days. Immunohistochemical labeling at 3 days for platelet endothelial cell adhesion molecule (PECAM), neuron-glial antigen 2 (NG2), lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), and class III β-tubulin identified endothelial cells, pericytes, lymphatics, and nerves, respectively. Media supplemented with bFGF or VEGF induced an increase in endothelial cell sprouting off existing vessels. Endothelial cell sprouting in both growth factor groups was inhibited by targeting pericytes with NG2 functional blocking antibody. VEGF caused an increase in the number of lymphatic/blood endothelial cell connections compared with media alone or bFGF groups. Finally, the comparison of the same network before and after angiogenesis stimulated by the supplement of media with 20% serum identified the ability of disconnected endothelial segments to reconnect to nearby vessels. The results establish a novel in situ angiogenesis model for investigating the location of capillary sprouting within an intact network, the role of pericytes, lymphatic/blood endothelial cell interactions, and the fate of specific endothelial cell segments. The rat mesentery culture system offers a unique tool for understanding the complex dynamics associated with angiogenesis in an intact adult tissue.

Differentiation Patterns of Embryonic Stem Cells in Two- versus Three-Dimensional Culture

Pluripotent stem cells are attractive candidates as a cell source for regenerative medicine and tissue engineering therapies. Current methods of differentiation result in low yields and impure populations of target phenotypes, with attempts for improved efficiency often comparing protocols that vary multiple parameters. This basic science study focused on a single variable to understand the effects of two-dimensional (2D) versus three-dimensional (3D) culture on directed differentiation. We compared mouse embryonic stem cells (ESCs) differentiated on collagen type I-coated surfaces (SLIDEs), embedded in collagen type I gels (GELs), and in suspension as embryoid bodies (EBs). For a systematic analysis in these studies, key parameters were kept identical to allow for direct comparison across culture configurations. We determined that all three configurations supported differentiation of ESCs and that the kinetics of differentiation differed greatly for cells cultured in 2D versus 3D. SLIDE cultures induced overall differentiation more quickly than 3D configurations, with earlier expression of cytoskeletal and extracellular matrix proteins. For 3D culture as GELs or EBs, cells clustered similarly, formed complex structures and promoted differentiation towards cardiovascular phenotypes. GEL culture, however, also allowed for contraction of the collagen matrix. For differentiation towards fibroblasts and smooth muscle cells which actively remodel their environment, GEL culture may be particularly beneficial. Overall, this study determined the effects of dimensionality on differentiation and helps in the rational design of protocols to generate phenotypes needed for tissue engineering and regenerative medicine.

Mesenchymal Stem Cells Overexpressing Ephrin-B2 Rapidly Adopt an Early Endothelial Phenotype with Simultaneous Reduction of Osteogenic Potential

Restoration of the vascular supply to ischemic tissues is of high clinical relevance, and proangiogenic therapies aim to reduce morbidity and mortality rates associated with the onset of cardiovascular disease. Stem cell therapy has been proposed as a potentially useful proangiogenic therapy. Mesenchymal stem cells (MSCs) have been shown to be proangiogenic and produce a number of cytokines involved in vessel development and maturation. Preclinical studies have reported increased angiogenesis after MSC delivery to the heart, and similar outcomes have been reported in recent clinical trials. Stem-cell-mediated neovascularization has been augmented by genetic modification with overexpression of angiogenic cytokines, including vascular endothelial growth factor (VEGF) and platelet-derived growth factor, showing promising results. In this study we aimed to enhance the proangiogenic capability of MSCs. MSCs were genetically modified to overexpress a versatile molecule, Ephrin-B2, involved in tissue morphogenesis and vascular development to enhance inherent neovascularization potential. Using nucleofection, Ephrin-B2 was transiently overexpressed on the cell surface of MSCs to recapitulate embryonic signaling and promote neovascularization. Ephrin-B2-expressing MSCs adopted an early endothelial phenotype under endothelial cell culture conditions increasing expression of von Willebrand factor and VEGF-Receptor 2. The cells had an increased ability to form vessel-like structures, produce VEGF, and incorporate into newly formed endothelial cell structures. These data indicate that MSCs expressing Ephrin-B2 represent a novel proangiogenic cell source to promote neovascularization in ischemic tissues.

Effects of shear stress on germ lineage specification of embryonic stem cells

Mechanobiology to date has focused on differentiated cells or progenitors, yet the effects of mechanical forces on early differentiation of pluripotent stem cells are still largely unknown. To study the effects of cellular deformation, we utilize a fluid flow bioreactor to apply steady laminar shear stress to mouse embryonic stem cells (ESCs) cultured on a two dimensional surface. Shear stress was found to affect pluripotency, as well as germ specification to the mesodermal, endodermal, and ectodermal lineages, as indicated by gene expression of OCT4, T-BRACHY, AFP, and NES, respectively. The ectodermal and mesodermal response to shear stress was dependent on stress magnitude (ranging from 1.5 to 15 dynes cm(-2)). Furthermore, increasing the duration from one to four days resulted in a sustained increase in T-BRACHY and a marked suppression of AFP. These changes in differentiation occurred concurrently with the activation of Wnt and estrogen pathways, as determined by PCR arrays for signalling molecules. Together these studies show that the mechanical microenvironment may be an important regulator during early differentiation events, including gastrulation. This insight furthers understanding of normal and pathological events during development, as well as facilitates strategies for scale up production of stem cells for clinical therapies.

Cytoskeletal Expression and Remodeling in Pluripotent Stem Cells.

Many emerging cell-based therapies are based on pluripotent stem cells, though complete understanding of the properties of these cells is lacking. In these cells, much is still unknown about the cytoskeletal network, which governs the mechanoresponse. The objective of this study was to determine the cytoskeletal state in undifferentiated pluripotent stem cells and remodeling with differentiation. Mouse embryonic stem cells (ESCs) and reprogrammed induced pluripotent stem cells (iPSCs), as well as the original un-reprogrammed embryonic fibroblasts (MEFs), were evaluated for expression of cytoskeletal markers. We found that pluripotent stem cells overall have a less developed cytoskeleton compared to fibroblasts. Gene and protein expression of smooth muscle cell actin, vimentin, lamin A, and nestin were markedly lower for ESCs than MEFs. Whereas, iPSC samples were heterogeneous with most cells expressing patterns of cytoskeletal proteins similar to ESCs with a small subpopulation similar to MEFs. This indicates that dedifferentiation during reprogramming is associated with cytoskeletal remodeling to a less developed state. In differentiation studies, it was found that shear stress-mediated differentiation resulted in an increase in expression of cytoskeletal intermediate filaments in ESCs, but not in iPSC samples. In the embryoid body model of spontaneous differentiation of pluripotent stem cells, however, both ESCs and iPSCs had similar gene expression for cytoskeletal proteins during early differentiation. With further differentiation, however, gene levels were significantly higher for iPSCs compared to ESCs. These results indicate that reprogrammed iPSCs more readily reacquire cytoskeletal proteins compared to the ESCs that need to form the network de novo. The strategic selection of the parental phenotype is thus critical not only in the context of reprogramming but also the ultimate functionality of the iPSC-differentiated cell population. Overall, this increased characterization of the cytoskeleton in pluripotent stem cells will allow for the better understanding and design of stem cell-based therapies.

Lack of vimentin impairs endothelial differentiation of embryonic stem cells.

The cytoskeletal filament vimentin is inherent to the endothelial phenotype and is critical for the proper function of endothelial cells in adult mice. It is unclear, however, if the presence of vimentin is necessary during differentiation to the endothelial phenotype. Here we evaluated gene and protein expression of differentiating wild type embryonic stem cells (WT ESCs) and vimentin knockout embryonic stem cells (VIM −/− ESCs) using embryoid bodies (EBs) formed from both cell types. Over seven days of differentiation VIM −/− EBs had altered morphology compared to WT EBs, with a rippled outer surface and a smaller size due to decreased proliferation. Gene expression of pluripotency markers decreased similarly for EBs of both cell types; however, VIM −/− EBs had impaired differentiation towards the endothelial phenotype. This was quantified with decreased expression of markers along the specification pathway, specifically the early mesodermal marker Brachy-T, the lateral plate mesodermal marker FLK1, and the endothelial-specific markers TIE2, PECAM, and VE-CADHERIN. Taken together, these results indicate that the absence of vimentin impairs spontaneous differentiation of ESCs to the endothelial phenotype in vitro.

Actin and myosin II modulate differentiation of pluripotent stem cells

Use of stem cell-based therapies in tissue engineering and regenerative medicine is hindered by efficient means of directed differentiation. For pluripotent stem cells, an initial critical differentiation event is specification to one of three germ lineages: endoderm, mesoderm, and ectoderm. Differentiation is known to be regulated by numerous extracellular and intracellular factors, but the role of the cytoskeleton during specification, or early differentiation, is still unknown. In these studies, we used agonists and antagonists to modulate actin polymerization and the actin-myosin molecular motor during spontaneous differentiation of embryonic stem cells in embryoid bodies. We found that inhibiting either actin polymerization or actin-myosin interactions led to a decrease in differentiation to the mesodermal lineage and an increase in differentiation to the endodermal lineage. Thus, targeting processes that regulate cytoskeletal tension may be effective in enhancing or inhibiting differentiation towards cells of the endodermal or mesodermal lineages, which include hepatocytes, islets, cardiomyocytes, endothelial cells, and osteocytes. Therefore, these fundamental findings demonstrate that modulation of the cytoskeleton may be useful in production for a range of cell-based therapies, including for liver, pancreatic, cardiac, vascular, and orthopedic applications.

Applying Shear Stress to Pluripotent Stem Cells

Thorough understanding of the effects of shear stress on stem cells is critical for the rationale design of large-scale production of cell-based therapies. This is of growing importance as emerging tissue engineering and regenerative medicine applications drive the need for clinically relevant numbers of both pluripotent stem cells (PSCs) and cells derived from PSCs. Here, we describe the use of a custom parallel plate bioreactor system to impose fluid shear stress on a layer of PSCs adhered to protein-coated glass slides. This system can be useful both for basic science studies in mechanotransduction and as a surrogate model for bioreactors used in large-scale production.

Proliferation of Cells From a Mouse Model of Regeneration

The ability to induce limb regeneration in humans is of growing interest in the field of regenerative medicine, particularly due to the increased number of amputees among military veterans. Unfortunately, mammals have limited regenerative capabilities as compared to amphibians, which can re-establish complex structures after traumatic injury. There have been a few clinically documented cases of digit regeneration in children [1], indicating that the potential to regenerate is not completely absent in humans. Mammalian models of epimorphic regeneration is primarily limited to the mouse digit, which has a level-specific response in that amputation at the terminal phalangeal element (P3) results in regeneration, but not at the next more proximal joint (P2) (Figure 1). Recently primary stromal cells were isolated from each of these mouse joints (P3 and P2, respectively) [2], which provides a unique opportunity to utilize in vitro techniques to identify differences in one of the phenotypes prevalent at the amputation plane of a regenerating and non-regenerating region.Copyright