Mending the Failing Heart with a Vascularized Cardiac Patch

Functional, stem-cell-containing cardiac grafts will require vascularized myocardial constructs to support their survival and integration into the host vasculature. Recently in Tissue Engineering, Part A, Lesman et al. (2009) reported the successful integration of vascular cells and hESC-derived cardiomyoctyes into stable grafts in rat recipients

Lymphatic Tissue Engineering and Regeneration

Abstract The lymphatic system is a major circulatory system within the body, responsible for the transport of interstitial fluid, waste products, immune cells, and proteins. Compared to other physiological systems, the molecular mechanisms and underlying disease pathology largely remain to be understood which has hindered advancements in therapeutic options for lymphatic disorders. Dysfunction of the lymphatic system is associated with a wide range of disease phenotypes and has also been speculated as a route to rescue healthy phenotypes in areas including cardiovascular disease, metabolic syndrome, and neurological conditions. This review will discuss lymphatic system functions and structure, cell sources for regenerating lymphatic vessels, current approaches for engineering lymphatic vessels, and specific therapeutic areas that would benefit from advances in lymphatic tissue engineering and regeneration

Hyaluronic Acid Hydrogels Support Cord-Like Structures from Endothelial Colony-Forming Cells

The generation of functional vascular networks has the potential to improve treatment for vascular diseases and to facilitate successful organ transplantation. Endothelial colony-forming cells (ECFCs) have robust proliferative potential and can form vascular networks in vivo. ECFCs are recruited from a bone marrow niche to the site of vascularization, where cues from the extracellular matrix instigate vascular morphogenesis. Although this process has been elucidated using natural matrix, little is known about vascular morphogenesis by ECFCs in synthetic matrix, a xeno-free scaffold that can provide a more controllable and clinically relevant alternative for regenerative medicine. We sought to study hyaluronic acid (HA) hydrogels as three-dimensional scaffolds for capillary-like structure formation from ECFCs, and to determine the crucial parameters needed to design such synthetic scaffolds. We found that ECFCs express HA-specific receptors and that vascular endothelial growth factor stimulates hyaluronidase expression in ECFCs. Using a well-defined and controllable three-dimensional HA culture system, we were able to decouple the effect of matrix viscoelasticity from changes in adhesion peptide density. We determined that decreasing matrix viscoelasticity, which corresponds to a loose ultrastructure, significantly increases ECFC vascular tube length and area, and that the effect of local delivery of vascular endothelial growth factor within the hydrogel depends on the makeup of the synthetic environment. Collectively, these results set forth initial design criteria that need to be considered in developing vascularized tissue constructs

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

Nuclear shaping during hESCs undergoing differentiation.

<p>A. Ensemble-averaged nuclear shape factors of undifferentiated hESCs and cells undergoing differentiation, as well as iPSCs, parental HLFs, HUVECs, and HFFs. The shape factor is close to zero for a highly elongated nucleus and unity for a perfectly round nucleus. B. Distributions of nuclear shape factors in undifferentiated hESCs and cells undergoing differentiation. At least 200 cells were probed in triplicate. C. Fraction of multi-lobulated hESCs, with a nucleus featuring at least one lobe (black curve), and showing no actin cap (red curve) as a function of days following onset of differentiation. D. Typical shapes of nuclei in undifferentiated hESCs (left panel) and cells 10 days after onset of differentiation (right panel). Scale bar, 100 µm. E. Distributions of nuclear shape factors in iPSCs, their parental HLFs, HUVECs, and HFFs. F. Fractions of multi-lobulated nuclei in undifferentiated hESCs, hESCs undergoing differentiation, iPSCs, their parental HLFs, HUVECs, and HFFs. *: P<0.05; **: P<0.01.</p

Status of Lamin A/C and LINC complexes in hESCs and iPSC during differentiation.

<p>A–D. Low- (10×, top panels) and high-magnification (60×, bottom panels) views of the organization of lamin A/C (A) and LINC complex components Nesprin2 giant (B), Nesprin3 (C), and Sun2 (D) at or near the nuclear envelope in undifferentiated hESCs and hESCs one and two days after switching to differentiation conditions. Cells were stained for nuclear DNA (DAPI), actin, TRA-1-81, and with antibodies against human lamin A/C, Nesprin2 giant, Nesprin3, and Sun2, as indicated, and visualized by immunofluorescence microscopy. Scale bar for 10× micrographs, 100 µm; Scale bar for 60× micrographs, 20 µm. E–G. Immunofluorescence micrographs showing the organization of actin and lamin A/C (E), LINC complex components Nesprin2 giant (F) and Sun2 (G) at the nuclear envelope of parental HLFs (top panels) and IPSCs (bottom panels). Scale bar, 20 µm.</p

The perinuclear actin cap progressively forms in hESCs following onset of differentiation.

<p>A–H. Status of apical perinuclear actin cap (A–D) and basal stress fibers (E–H) in undifferentiated hESCs at day 0 (A and E), as well as two (B and F), five (C and G), and ten days (D and G) after induction of differentiation (+VEGF and collagen IV). Red, purple, and orange arrows indicate examples of cells showing no actin cap, a disrupted/disorganized actin cap, and a well-organized actin cap, respectively. Cells were stained for differentiation marker TRA-1-81, nuclear DNA (DAPI), and actin. Scale bar, 20 µm. I. Evolution of the fraction of TRA-1-81-negative hESCs showing an actin cap. No actin cap was present in TRA-1-81-positive hESCs. J. Proportion of TRA-1-81-positive and TRA-1-81-negative hESCs showing an organized actin cap, a disorganized actin cap, or no actin cap, two and five days after onset of differentiation. K. Distribution of cells showing an actin cap in hESCs after 10 days of differentiation compared to HLFs. At least 100 cells in triplicate for a total of 300 cells were probed per condition.</p

Lamin A/C is required for the proper differentiation of mouse embryonic stem cells (mESCs).

<p>A. Representative micrographs of basal (left columns) and apical (right columns) actin of <i>Lmna^+/+</i>, <i>Lmna^+/−</i>, and <i>Lmna^−/−</i> mouse embryonic stem cells at day 0 (top row), at 3 days of differentiation (middle row), and after 14 days of differentiation (bottom row), illustrating the earlier appearance of the actin cap in wildtype cells (by 3 days) when compared to heterozygotes (∼7 days) and knockouts (>14 days). All scale bars: 20 µm. B. Flow cytometry analysis of normalized stage-specific embryonic antigen 1 (SSEA1) levels of <i>Lmna^+/+</i>, <i>Lmna^+/−</i>, and <i>Lmna^−/−</i> mESCs through 14 days of differentiation. C. Replating efficiencies of <i>Lmna^+/+</i>, <i>Lmna^+/−</i>, and <i>Lmna^−/−</i> mESCs after 3, 7, and 14 days of differentiation. *: P<0.05; **: P<0.01. ***: P<0.001; ns: non-significant difference.</p