Sravanti Kusuma

Functional neovascularization of biodegradable dextran hydrogels with multiple angiogenic growth factors

Slow vascularization of functional blood limits the transplantation of tissue constructs and the recovery of ischemic and wounded tissues. Despite the widespread investigation of polysaccharide-based hydrogel scaffolds for their therapeutic applications, blood vessel ingrowth into these hydrogel scaffolds remains a challenge. We hypothesized that modifying the properties of biodegradable hydrogel scaffolds with immobilization of multiple angiogenic growth factors (GFs) would induce a rapid proliferation of functional vasculature into the scaffolds. To this end, we remodeled the hydrogel structure by decreasing crosslinking density via reduced degree of substitution of crosslinking groups, which resulted in improved hydrogel properties including reduced rigidity, increased swelling, increased vascular endothelial GF (VEGF) release capability, and facilitated rapid hydrogel disintegration and tissue ingrowth. Immobilizing VEGF in the scaffolds promoted tissue ingrowth and expedited biodegradation. Furthermore, a synergistic effect of multiple angiogenic GFs was established; the coimmobilization of VEGF+ angiopoietin-1, and VEGF+ insulin-like GF+ stromal cell-derived factor-1 induced more and larger blood vessels than any individual GF, while the combination of all GFs dramatically increased the size and number of newly formed functional vessels. Altogether, our data demonstrate that rapid, efficient, and functional neovascularization can be achieved by precisely manipulating hydrogel scaffold properties and immobilizing defined angiogenic GFs.

Self-organized vascular networks from human pluripotent stem cells in a synthetic matrix

The success of tissue regenerative therapies is contingent on functional and multicellular vasculature within the redeveloping tissue. Although endothelial cells (ECs), which compose the vasculature’s inner lining, are intrinsically able to form nascent networks, these structures regress without the recruitment of pericytes, supporting cells that surround microvessel endothelium. Reconstruction of typical in vivo microvascular architecture traditionally has been done using distinct cell sources of ECs and pericytes within naturally occurring matrices; however, the limited sources of clinically relevant human cells and the inherent chemical and physical properties of natural materials hamper the translational potential of these approaches. Here we derived a bicellular vascular population from human pluripotent stem cells (hPSCs) that undergoes morphogenesis and assembly in a synthetic matrix. We found that hPSCs can be induced to codifferentiate into early vascular cells (EVCs) in a clinically relevant strategy amenable to multiple hPSC lines. These EVCs can mature into ECs and pericytes, and can self-organize to form microvascular networks in an engineered matrix. These engineered human vascular networks survive implantation, integrate with the host vasculature, and establish blood flow. This integrated approach, in which a derived bicellular population is exploited for its intrinsic self-assembly capability to create microvasculature in a deliverable matrix, has vast ramifications for vascular construction and regenerative medicine.

Reconstructing the differentiation niche of embryonic stem cells using biomaterials.

The biochemical cues and topographical architecture of the extracellular environment extensively influence ES cell fate. The microenvironment surrounding the developing embryo presents these instructive cues in a complex and interactive manner in order to guide cell fate decisions. Current stem cell research aims to reconstruct this multifaceted embryonic niche to recapitulate development in vitro. This review focuses on 2D and 3D differentiation niches created from natural and synthetic biomaterials to guide the differentiation of ES cells toward specific lineages. Biomaterials engineered to present specific physical constraints are also reviewed for their role in differentiation.

Smooth-Muscle-Like Cells Derived from Human Embryonic Stem Cells Support and Augment Cord-Like Structures In Vitro

Engineering vascularized tissue is crucial for its successful implantation, survival, and integration with the host tissue. Vascular smooth muscle cells (v-SMCs) provide physical support to the vasculature and aid in maintaining endothelial viability. In this study, we show an efficient derivation of v-SMCs from human embryonic stem cells (hESCs), and demonstrate their functionality and ability to support the vasculature in vitro. Human ESCs were differentiated in monolayers and supplemented with platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-beta 1 (TGF-β1). Human ESC-derived smooth-muscle-like cells (SMLCs) were found to highly express specific smooth muscle cell (SMC) markers—including α-smooth muscle actin, calponin, SM22, and smooth muscle myosin heavy chain—to produce and secrete fibronectin and collagen, and to contract in response to carbachol. In vitro tubulogenesis assays revealed that these hESC-derived SMLCs interacted with human endothelial progenitor cell (EPCs) to form longer and thicker cord-like structures in vitro. We have demonstrated a simple protocol for the efficient derivation of highly purified SMLCs from hESCs. These in vitro functional SMLCs interacted with EPCs to support and augment capillary-like structures (CLSs), demonstrating the potential of hESCs as a cell source for therapeutic vascular tissue engineering.

Functional groups affect physical and biological properties of dextran-based hydrogels

Modification of dextran backbone allows the development of a hydrogel with specific characteristics. To enhance their functionality for tissue-engineered scaffolds, a series of dextran-based macromers was synthesized by incorporating various functional groups, including allyl isocyanate (Dex-AI), ethylamine (Dex-AE), chloroacetic acid (Dex-AC), or maleic-anhydride (Dex-AM) into dextrans. The dextran-based biodegradable hybrid hydrogels are developed by integrating polyethylene glycol diacrylate (PEGDA). To explore the effect of different derivatives on hydrogel properties, three different ratios of Dex/PEGDA are examined: low (20/80), medium (40/60), and high (60/40). Differences in physical and biological properties of the hydrogels are found, including swelling, degradation rate, mechanics, crosslinking density, biocompatibility (in vitro and in vivo), and vascular endothelial growth factor release. The results also indicate that the incorporation of amine groups into dextran gives rise to hydrogels with better biocompatible and release properties. We, therefore, conclude that the incorporation of different functional groups affects the fundamental properties of a dextran-based hydrogel network, and that amine groups are preferred to generate hydrogels for biomedical use.

Perivascular cells in blood vessel regeneration

Vascular engineering seeks to design and construct functional blood vessels comprising endothelial cells (ECs) and perivascular cells (PCs), with the ultimate goal of clinical translation. While EC behavior has been extensively investigated, PCs play an equally significant role in the development of novel regenerative strategies, providing functionality and stability to vessels. The two major classes of PCs are vascular smooth muscle cells (vSMCs) and pericytes; vSMCs can be further sub‐classified as either contractile or synthetic. The inclusion of these cell types is crucial for successful regeneration of blood vessels. Furthermore, understanding distinctions between vSMCs and pericytes will enable improved therapeutics in a tissue‐specific manner. Here we focus on the approaches and challenges facing the use of PCs in vascular regeneration, including their characteristics, stem cell sources, and interactions with ECs. Finally, we discuss biochemical and microRNA (miR) regulators of PC behavior and engineering approaches that mimic various cues affecting PC function.

The differential formation of the LINC-mediated perinuclear actin cap in pluripotent and somatic cells.

The actin filament cytoskeleton mediates cell motility and adhesion in somatic cells. However, whether the function and organization of the actin network are fundamentally different in pluripotent stem cells is unknown. Here we show that while conventional actin stress fibers at the basal surface of cells are present before and after onset of differentiation of mouse (mESCs) and human embryonic stem cells (hESCs), actin stress fibers of the actin cap, which wrap around the nucleus, are completely absent from undifferentiated mESCs and hESCs and their formation strongly correlates with differentiation. Similarly, the perinuclear actin cap is absent from human induced pluripotent stem cells (hiPSCs), while it is organized in the parental lung fibroblasts from which these hiPSCs are derived and in a wide range of human somatic cells, including lung, embryonic, and foreskin fibroblasts and endothelial cells. During differentiation, the formation of the actin cap follows the expression and proper localization of nuclear lamin A/C and associated linkers of nucleus and cytoskeleton (LINC) complexes at the nuclear envelope, which physically couple the actin cap to the apical surface of the nucleus. The differentiation of hESCs is accompanied by the progressive formation of a perinuclear actin cap while induced pluripotency is accompanied by the specific elimination of the actin cap, and that, through lamin A/C and LINC complexes, this actin cap is involved in progressively shaping the nucleus of hESCs undergoing differentiation. While, the localization of lamin A/C at the nuclear envelope is required for perinuclear actin cap formation, it is not sufficient to control nuclear shape.

Engineering blood vessels using stem cells: innovative approaches to treat vascular disorders.

Vascular disease is the leading cause of mortality in the USA, providing the impetus for new treatments and technologies. Current therapies rely on the implantation of stents or grafts to treat injured blood vessels. However, these therapies may be immunogenic or may incompletely recover the functional integrity of the vasculature. In light of these shortcomings, cell-based therapies provide new treatment options to heal damaged areas with more suitable substitutes. Current clinical trials employing stem cell-based therapies involve the transfusion of harvested endothelial progenitor cells. While the results from these trials have been encouraging, utilizing tissue-engineered approaches could yield technologically advanced solutions. This article discusses engineered stem cell-based therapies from three angles: the differentiation of adult stem cells, such as mesenchymal stem cells and endothelial progenitor cells, into vascular lineages; investigation of human embryonic stem cells and induced pluripotent stem cells as inexhaustible sources of vascular cells; and tissue-engineering approaches, which incorporate these vascular progenitor cells into biomimetic scaffolds to guide regeneration. The optimal solution to vascular disease lies at the interface of these technologies – embedding differentiated cells into engineered scaffolds to impart precise control over vascular regeneration.

The extracellular matrix is a novel attribute of endothelial progenitors and of hypoxic mature endothelial cells

Extracellular matrix (ECM) production is critical to preserve the function and integrity of mature blood vessels. Toward the engineering of blood vessels, studies have centered on ECM production by supporting cells, whereas few studies implicate endothelial cells (ECs) with ECM synthesis. Here, we elucidate variations between cultured human arterial, venous, and progenitor ECs with respect to ECM deposition assembly, composition, and response to biomolecular and physiological factors. Our studies reveal that progenitor ECs, endothelial colony‐forming cells (ECFCs), deposit collagen IV, fibronectin, and laminin that assemble to an organized weblike structure, as confirmed by decellularized cultures. Mature ECs only express these ECM proteins intracellularly. ECFC‐derived ECM is abrogated in response to TGFβ signaling inhibition and actin cytoskeleton disruption. Hypoxic (1%) and physiological (5%) O2 tension stimulate ECM deposition from mature ECs. Interestingly, deposition of collagen I is observed only under 5% O2 tension. ECM production from all ECs is found to be regulated by hypoxia‐inducible factors 1α and 2α but differentially in the different cell lines. Collectively, we suggest that ECM deposition and assembly by ECs is dependent on maturation stage and oxygen supply and that these findings can be harnessed to advance engineered vascular therapeutics.—Kusuma, S., Zhao, S., Gerecht, S. The extracellular matrix is a novel attribute of endothelial progenitors and of hypoxic mature endothelial cells. FASEB J. 26, 4925–4936 (2012). www.fasebj.org

Low Oxygen Tension Enhances Endothelial Fate of Human Pluripotent Stem Cells

Objective—A critical regulator of the developing or regenerating vasculature is low oxygen tension. Precise elucidation of the role of low oxygen environments on endothelial commitment from human pluripotent stem cells necessitates controlled in vitro differentiation environments. Approach and Results—We used a feeder-free, 2-dimensional differentiation system in which we could monitor accurately dissolved oxygen levels during human pluripotent stem cell differentiation toward early vascular cells (EVCs). We found that oxygen uptake rate of differentiating human pluripotent stem cells is lower in 5% O2 compared with atmospheric conditions. EVCs differentiated in 5% O2 had an increased vascular endothelial cadherin expression with clusters of vascular endothelial cadherin+ cells surrounded by platelet-derived growth factor &bgr;+ cells. When we assessed the temporal effects of low oxygen differentiation environments, we determined that low oxygen environments during the early stages of EVC differentiation enhance endothelial lineage commitment. EVCs differentiated in 5% O2 exhibited an increased expression of vascular endothelial cadherin and CD31 along with their localization to the membrane, enhanced lectin binding and acetylated low-density lipoprotein uptake, rapid cord-like structure formation, and increased expression of arterial endothelial cell markers. Inhibition of reactive oxygen species generation during the early stages of differentiation abrogated the endothelial inductive effects of the low oxygen environments. Conclusions—Low oxygen tension during early stages of EVC derivation induces endothelial commitment and maturation through the accumulation of reactive oxygen species, highlighting the importance of regulating oxygen tensions during human pluripotent stem cell-vascular differentiation.