Sepideh Heydarkhan-Hagvall

Three-dimensional electrospun ECM-based hybrid scaffolds for cardiovascular tissue engineering.

Electrospinning using natural proteins or synthetic polymers is a promising technique for the fabrication of fibrous scaffolds for various tissue engineering applications. However, one limitation of scaffolds electrospun from natural proteins is the need to cross-link with glutaraldehyde for stability, which has been postulated to lead to many complications in vivo including graft failure. In this study, we determined the characteristics of hybrid scaffolds composed of natural proteins including collagen and elastin, as well as gelatin, and the synthetic polymer poly(epsilon-caprolactone) (PCL), so to avoid chemical cross-linking. Fiber size increased proportionally with increasing protein and polymer concentrations, whereas pore size decreased. Electrospun gelatin/PCL scaffolds showed a higher tensile strength when compared to collagen/elastin/PCL constructs. To determine the effects of pore size on cell attachment and migration, both hybrid scaffolds were seeded with adipose-derived stem cells. Scanning electron microscopy and nuclei staining of cell-seeded scaffolds demonstrated the complete cell attachment to the surfaces of both hybrid scaffolds, although cell migration into the scaffold was predominantly seen in the gelatin/PCL hybrid. The combination of natural proteins and synthetic polymers to create electrospun fibrous structures resulted in scaffolds with favorable mechanical and biological properties.

Reprogrammed Mouse Fibroblasts Differentiate into Cells of the Cardiovascular and Hematopoietic Lineages

Forced expression of the four transcription factors Oct4, Sox2, c‐Myc, and Klf4 is sufficient to confer a pluripotent state upon the murine fibroblast genome, generating induced pluripotent stem (iPS) cells. Although the differentiation potential of these cells is thought to be equivalent to that of embryonic stem (ES) cells, it has not been rigorously determined. In this study, we sought to identify the capacity of iPS cells to differentiate into Flk1‐positive progenitors and their mesodermal progeny, including cells of the cardiovascular and hematopoietic lineages. Immunostaining of tissues from iPS cell‐derived chimeric mice demonstrated that iPS cells could contribute in vivo to cardiomyocytes, smooth muscle cells, endothelial cells, and hematopoietic cells. To compare the in vitro differentiation potential of murine ES and iPS cells, we either induced embryoid body (EB) formation of each cell type or cultured the cells on collagen type IV (ColIV), an extracellular matrix protein that had been reported to direct murine ES cell differentiation to mesodermal lineages. EB formation and exposure to ColIV both induced iPS cell differentiation into cells that expressed cardiovascular and hematopoietic markers. To determine whether ColIV‐differentiated iPS cells contained a progenitor cell with cardiovascular and hematopoietic differentiation potential, Flk1‐positive cells were isolated by magnetic cell sorting and exposed to specific differentiation conditions, which induced differentiation into functional cardiomyocytes, smooth muscle cells, endothelial cells, and hematopoietic cells. Our data demonstrate that murine iPS cells, like ES cells, can differentiate into cells of the cardiovascular and hematopoietic lineages and therefore may represent a valuable cell source for applications in regenerative medicine.

Human Adipose Stem Cells: A Potential Cell Source for Cardiovascular Tissue Engineering

Background/Aims: A crucial step in providing clinically relevant applications of cardiovascular tissue engineering involves the identification of a suitable cell source. The objective of this study was to identify the exogenous and endogenous parameters that are critical for the differentiation of human adipose stem cells (hASCs) into cardiovascular cells. Methods: hASCs were isolated from human lipoaspirate samples, analyzed, and subjected to two differentiation protocols. Results: As shown by fluorescence-activated cell sorter (FACS) analysis, a population of hASCs expressed stem cell markers including CXCR4, CD34, c-kit, and ABCG2. Further, FACS and immunofluorescence analysis of hASCs, cultured for 2 weeks in DMEM-20%-FBS, showed the expression of smooth muscle cell (SMC)-specific markers including SM α-actin, basic calponin, h-caldesmon and SM myosin. hASCs, cultured for 2 weeks in endothelial cell growth medium-2 (EGM-2), formed a network of branched tube-like structures positive for CD31, CD144, and von Willebrand factor. The frequency of endothelial cell (EC) marker-expressing cells was passage number-dependent. Moreover, hASCs attached and formed a confluent layer on top of electrospun collagen-elastin scaffolds. Scanning electron microscopy and DAPI staining confirmed the integration of hASCs with the fibers and formation of a cell-matrix network. Conclusion: Our results indicate that hASCs are a potential cell source for cardiovascular tissue engineering; however, the differentiation capacity of hASCs into SMCs and ECs is passage number- and culture condition-dependent.

Collagen IV Induces Trophoectoderm Differentiation of Mouse Embryonic Stem Cells

The earliest segregation of lineages in the developing embryo is the commitment of cells to the inner cell mass or the trophoectoderm in preimplantation blastocysts. The exogenous signals that control commitment to a particular cell lineage are poorly understood; however, it has been suggested that extracellular “niche” and extracellular matrix, in particular, play an important role in determining the developmental fate of stem cells. Collagen IV (ColIV) has been reported to direct embryonic stem (ES) cell differentiation to mesodermal lineages in both mouse and human ES cells. To define the effects of ColIV on ES cell differentiation and to identify the resulting heterogeneous cell types, we performed microarray analyses and determined global gene expression. We observed that ColIV induced the expression of mesodermal genes specific to hematopoietic, endothelial, and smooth muscle cells and, surprisingly, also a panel of trophoectoderm‐restricted markers. This effect was specific to collagen IV, as no trophoblast differentiation was seen on collagen I, laminin, or fibronectin. Stimulation with basic fibroblast growth factor (FGF) or FGF4 increased the number of trophoectodermal cells. These cells were isolated under clonal conditions and successfully differentiated into a variety of trophoblast derivatives. Interestingly, differentiation of ES cells to trophoblastic lineages was only seen in ES cell lines maintained on embryonic feeder layers and was caudal‐type homeobox protein 2 (Cdx2)‐dependent, consistent with Cdx2s postulated role in trophoectoderm commitment. Our data suggest that, given the appropriate extracellular stimuli, mouse embryonic stem cells can differentiate into trophoectoderm.

The use of three-dimensional nanostructures to instruct cells to produce extracellular matrix for regenerative medicine strategies

Synthetic polymers or naturally-derived extracellular matrix (ECM) proteins have been used to create tissue engineering scaffolds; however, the need for surface modification in order to achieve polymer biocompatibility and the lack of biomechanical strength of constructs built using proteins alone remain major limitations. To overcome these obstacles, we developed novel hybrid constructs composed of both strong biosynthetic materials and natural human ECM proteins. Taking advantage of the ability of cells to produce their own ECM, human foreskin fibroblasts were grown on silicon-based nanostructures exhibiting various surface topographies that significantly enhanced ECM protein production. After 4 weeks, cell-derived sheets were harvested and histology, immunochemistry, biochemistry and multiphoton imaging revealed the presence of collagens, tropoelastin, fibronectin and glycosaminoglycans. Following decellularization, purified sheet-derived ECM proteins were mixed with poly(epsilon-caprolactone) to create fibrous scaffolds using electrospinning. These hybrid scaffolds exhibited excellent biomechanical properties with fiber and pore sizes that allowed attachment and migration of adipose tissue-derived stem cells. Our study represents an innovative approach to generate strong, non-cytotoxic scaffolds that could have broad applications in tissue regeneration strategies.

Influence of systematically varied nano-scale topography on cell morphology and adhesion

The types of cell–matrix adhesions and the signals they transduce strongly affect the cell-phenotype. We hypothesized that cells sense and respond to the three-dimensionality of their environment, which could be modulated by nano-structures on silicon surfaces. Human foreskin fibroblasts were cultured on nano-structures with different patterns (nano-post and nano-grate) and heights for 3 days. The presence of integrin α5, β1, β3, paxillin and phosphorylated FAK (pFAK) were detected by western blot and immunofluorescence. Integrin β3 exhibited stronger signals on nano-grates. pFAK and paxillin were observed as small dot-like patterns on the cell-periphery on nano-posts and as elongated and aligned patterns on nano-grates. Collectively, our observations highlighted the presence of focal (integrin β1, β3, pFAK, paxillin), fibrillar (integrin α5, β1) and 3-D matrix (integrin α5, β1, paxillin) adhesions on nano-structures. The presented nano-structures offer interesting opportunities to study the interaction of cells with topographical features comparable to the size of extracellular matrix components.

The effect of vitronectin on the differentiation of embryonic stem cells in a 3D culture system

While stem cell niches in vivo are complex three-dimensional (3D) microenvironments, the relationship between the dimensionality of the niche to its function is unknown. We have created a 3D microenvironment through electrospinning to study the impact of geometry and different extracellular proteins on the development of cardiac progenitor cells (Flk-1(+)) from resident stem cells and their differentiation into functional cardiovascular cells. We have investigated the effect of collagen IV, fibronectin, laminin and vitronectin on the adhesion and proliferation of murine ES cells as well as the effects of these proteins on the number of Flk-1(+) cells cultured in 2D conditions compared to 3D system in a feeder free condition. We found that the number of Flk-1(+) cells was significantly higher in 3D scaffolds coated with laminin or vitronectin compared to colIV-coated scaffolds. Our results show the importance of defined culture systems in vitro for studying the guided differentiation of pluripotent embryonic stem cells in the field of cardiovascular tissue engineering and regenerative medicine.

Endothelial differentiation in multipotent cells derived from mouse and human white mature adipocytes

White mature adipocytes give rise to multipotent cells, so-called de-differentiated fat (DFAT) cells, when losing their fat in culture. The objective of this study was to examine the ability of DFAT cells to give rise to endothelial cells (ECs) in vitro and vivo. We demonstrate that mouse and human DFAT cells, derived from adipose tissue and lipospirate, respectively, initially lack expression of CD34, CD31, CD146, CD45 and pericyte markers, distinguishing them from progenitor cells previously identified in adipose stroma. The DFAT cells spontaneously differentiate into vascular ECs in vitro, as determined by real-time PCR, fluorescence activated cell sorting, immunostaining, and formation of tube structures. Treatment with bone morphogenetic protein (BMP)4 and BMP9, important in regulating angiogenesis, significantly enhances the EC differentiation. Furthermore, adipocyte-derived cells from Green Fluorescent Protein-transgenic mice were detected in the vasculature of infarcted myocardium up to 6 weeks after ligation of the left anterior descending artery in mice. We conclude that adipocyte-derived multipotent cells are able to spontaneously give rise to ECs, a process that is promoted by BMPs and may be important in cardiovascular regeneration and in physiological and pathological changes in fat and other tissues.

Cell growth as a sheet on three-dimensional sharp-tip nanostructures

Cells in vivo encounter with and react to the extracellular matrix materials on a nanometer scale. Recent advances in nanofabrication technologies allowing the precise control of a nanostructures pattern, periodicity, shape, and height have enabled a systematic study of cell interactions with three-dimensional nanotopographies. In this report, we examined the behavior of human foreskin fibroblasts on well-ordered dense arrays (post and grate patterns with a 230-nm pitch) of sharp-tip nanostructures with varying three-dimensionalities (from 50 to 600 nm in structural height) over time-until a cell sheet was formed. Although cells started out smaller and proliferated slower on tall nanostructures (both posts and grates) than on smooth surfaces, they became confluent to form a sheet in 3 weeks. On grate patterns, significant cell elongation in alignment with the underlying pattern was observed and maintained over time. On tall nanostructures, cells grew while raised on sharp tips, resulting in a weak total adherence to the solid surface. A sheet of cells was easily peeled off from such surfaces, suggesting that nanoscale topographies can be used as the basis for cell-sheet tissue engineering.

Manual isolation of adipose-derived stem cells from human lipoaspirates.

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.