Pluripotent stem cell-derived committed cardiac progenitors remuscularize damaged ischemic hearts and improve their function in pigs.

Ischemic heart disease, which is often associated with irreversibly damaged heart muscle, is a major global health burden. Here, we report the potential of stem cell-derived committed cardiac progenitors (CCPs) have in regenerative cardiology. Human pluripotent embryonic stem cells were differentiated to CCPs on a laminin 521 + 221 matrix, characterized with bulk and single-cell RNA sequencing, and transplanted into infarcted pig hearts. CCPs differentiated for eleven days expressed a set of genes showing higher expression than cells differentiated for seven days. Functional heart studies revealed significant improvement in left ventricular ejection fraction at four and twelve weeks following transplantation. We also observed significant improvements in ventricular wall thickness and a reduction in infarction size after CCP transplantation (p-value < 0.05). Immunohistology analyses revealed in vivo maturation of the CCPs into cardiomyocytes (CM). We observed temporary episodes of ventricular tachyarrhythmia (VT) in four pigs and persistent VT in one pig, but the remaining five pigs exhibited normal sinus rhythm. Importantly, all pigs survived without the formation of any tumors or VT-related abnormalities. We conclude that pluripotent stem cell-derived CCPs constitute a promising possibility for myocardial infarction treatment and that they may positively impact regenerative cardiology.

Viral proteases activate the CARD8 inflammasome in the human cardiovascular system.

Nucleotide-binding oligomerization domain (NBD), leucine-rich repeat (LRR) containing protein family (NLRs) are intracellular pattern recognition receptors that mediate innate immunity against infections. The endothelium is the first line of defense against blood-borne pathogens, but it is unclear which NLRs control endothelial cell (EC) intrinsic immunity. Here, we demonstrate that human ECs simultaneously activate NLRP1 and CARD8 inflammasomes in response to DPP8/9 inhibitor Val-boro-Pro (VbP). Enterovirus Coxsackie virus B3 (CVB3)-the most common cause of viral myocarditis-predominantly activates CARD8 in ECs in a manner that requires viral 2A and 3C protease cleavage at CARD8 p.G38 and proteasome function. Genetic deletion of CARD8 in ECs and human embryonic stem cell-derived cardiomyocytes (HCMs) attenuates CVB3-induced pyroptosis, inflammation, and viral propagation. Furthermore, using a stratified endothelial-cardiomyocyte co-culture system, we demonstrate that deleting CARD8 in ECs reduces CVB3 infection of the underlying cardiomyocytes. Our study uncovers the unique role of CARD8 inflammasome in endothelium-intrinsic anti-viral immunity.

Evaluating Capture Sequence Performance for Single-Cell CRISPR Activation Experiments.

The combination of single-cell RNA sequencing with CRISPR inhibition/activation provides a high-throughput approach to simultaneously study the effects of hundreds if not thousands of gene perturbations in a single experiment. One recent development in CRISPR-based single-cell techniques introduces a feature barcoding technology that allows for the simultaneous capture of mRNA and guide RNA (gRNA) from the same cell. This is achieved by introducing a capture sequence, whose complement can be incorporated into each gRNA and that can be used to amplify these features prior to sequencing. However, because the technology is in its infancy, there is little information available on how such experimental parameters can be optimized. To overcome this, we varied the capture sequence, capture sequence position, and gRNA backbone to identify an optimal gRNA scaffold for CRISPR activation gene perturbation studies. We provide a report on our screening approach along with our observations and recommendations for future use.

Laminins in Cellular Differentiation.

Basement membrane laminins (LNs) have been shown to modulate cellular phenotypes and differentiation both in vitro and during organogenesis in vivo. At least 16 laminin isoforms are present in mammals, and most are available as recombinant proteins. Ubiquitous LN511 and LN521 promote the clonal derivation and expansion of pluripotent embryonic stem cells (ESCs), and, together with other highly cell type-specific laminins, they can support the differentiation of stem cells into, for example, cardiac muscle fibers, retinal pigmented epithelial (RPE) cells and photoreceptors, dopamine (DA) neurons, and skin keratinocytes. The laminin-supported differentiation methods are highly reproducible and can be made chemically defined and fully xeno-free - a prerequisite for preparing therapeutic stem cell-derived cells. In this review we describe recent work on the use of laminin-based cell culture matrices in stem cell differentiation.

In Vivo Generation of Post-infarct Human Cardiac Muscle by Laminin-Promoted Cardiovascular Progenitors.

Regeneration of injured human heart muscle is limited and an unmet clinical need. There are no methods for the reproducible generation of clinical-quality stem cell-derived cardiovascular progenitors (CVPs). We identified laminin-221 (LN-221) as the most likely expressed cardiac laminin. We produced it as human recombinant protein and showed that LN-221 promotes differentiation of pluripotent human embryonic stem cells (hESCs) toward cardiomyocyte lineage and downregulates pluripotency and teratoma-associated genes. We developed a chemically defined, xeno-free laminin-based differentiation protocol to generate CVPs. We show high reproducibility of the differentiation protocol using time-course bulk RNA sequencing developed from different hESC lines. Single-cell RNA sequencing of CVPs derived from hESC lines supported reproducibility and identified three main progenitor subpopulations. These CVPs were transplanted into myocardial infarction mice, where heart function was measured by echocardiogram and human heart muscle bundle formation was identified histologically. This method may provide clinical-quality cells for use in regenerative cardiology.

Immobilization of vitronectin-binding heparan sulfates onto surfaces to support human pluripotent stem cells.

Functionalizing medical devices with polypeptides to enhance their performance has become important for improved clinical success. The extracellular matrix (ECM) adhesion protein vitronectin (VN) is an effective coating, although the chemistry used to attach VN often reduces its bioactivity. In vivo, VN binds the ECM in a sequence-dependent manner with heparan sulfate (HS) glycosaminoglycans. We reasoned therefore that sequence-based affinity chromatography could be used to isolate a VN-binding HS fraction (HS9) for use as a coating material to capture VN onto implant surfaces. Binding avidity and specificity of HS9 were confirmed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR)-based assays. Plasma polymerization of allylamine (AA) to tissue culture-treated polystyrene (TCPS) was then used to capture and present HS9 as determined by radiolabeling and ELISA. HS9-coated TCPS avidly bound VN, and this layered surface supported the robust attachment, expansion, and maintenance of human pluripotent stem cells. Compositional analysis demonstrated that 6-O- and N-sulfation, as well as lengths greater than three disaccharide units (dp6) are critical for VN binding to HS-coated surfaces. Importantly, HS9 coating reduced the threshold concentration of VN required to create an optimally bioactive surface for pluripotent stem cells. We conclude that affinity-purified heparan sugars are able to coat materials to efficiently bind adhesive factors for biomedical applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1887-1896, 2018.

Defining a threshold surface density of vitronectin for the stable expansion of human embryonic stem cells.

Current methodology for pluripotent human embryonic stem cells (hESCs) expansion relies on murine sarcoma basement membrane substrates (Matrigel™), which precludes the use of these cells in regenerative medicine. To realize the clinical efficacy of hESCs and their derivatives, expansion of these cells in a defined system that is free of animal components is required. This study reports the successful propagation of hESCs (HES-3 and H1) for > 20 passages on tissue culture-treated polystyrene plates, coated from 5 µg/mL of human plasma-purified vitronectin (VN) solution. Cells maintain expression of pluripotent markers Tra1-60 and OCT-4 and are karyotypically normal after 20 passages of continuous culture. In vitro and in vivo differentiation of hESC by embryoid body formation and teratoma yielded cells from the ecto-, endo-, and mesoderm lineages. VN immobilized on tissue culture polystyrene was characterized using a combination of X-ray photoemission spectroscopy, atomic force microscopy, and quantification of the VN surface density with a Bradford protein assay. Ponceau S staining was used to measure VN adsorption and desorption kinetics. Tuning the VN surface density, via the concentration of depositing solution, revealed a threshold surface density of 250 ng/cm², which is required for hESCs attachment, proliferation, and differentiation. Cell attachment and proliferation assays on VN surface densities above this threshold show the substrate properties to be equally viable.

Impact of vitronectin concentration and surface properties on the stable propagation of human embryonic stem cells.

The standard method for culturing human embryonic stem cells (hESC) uses supporting feeder layers of cells or an undefined substrate, Matrigel(™), which is a basement membrane extracted from murine sarcoma. For stem cell therapeutic applications, a superior alternative would be a defined, artificial surface that is based on immobilized human plasma vitronectin (VN), which is an adhesion-mediating protein. Therefore, VN adsorbed to diverse polymer surfaces was explored for the continuous propagation of hESC. Cells propagated on VN-coated tissue culture polystyrene (TCPS) are karyotypically normal after >10 passages of continuous culture, and are able to differentiate into embryoid bodies containing all three germ layers. Expansion rates and pluripotent marker expression verified that a minimal VN surface density threshold is required on TCPS. Further exploration of adsorbed VN was conducted on polymer substrates with different properties, ranging from hydrophilic to hydrophobic and including cationic and anionic polyelectrolyte coatings. Despite differing surface properties, these substrates adsorbed VN above the required surface density threshold and were capable of supporting hESC expansion for >10 passages. Correlating wettability of the VN-coated surfaces with the response of cultured hESC, higher cell expansion rates and OCT-4 expression levels were found for VN-coated TCPS, which exhibits a water contact angle close to 65°. Importantly, this simple, defined surface matches the performance of the benchmark Matrigel, which is a hydrogel with highly complex composition.