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.

Postnatal deletion of Spns2 prevents neuroinflammation without compromising blood vascular functions.

Protein Spinster homolog 2 (Spns2) is a sphingosine-1-phosphate (S1P) transporter that releases S1P to regulate lymphocyte egress and trafficking. Global deletion of Spns2 (Spns2-/-) has been shown to reduce disease severity in several autoimmune disease models. To examine whether Spns2 could be exploited as a drug target, we generated and characterized the mice with postnatal knockout of Spns2 (Spns2-Mx1Cre). Our results showed that Spns2-Mx1Cre mice had significantly low number of lymphocytes in blood and lymphoid organs similar to Spns2-/- mice. Lymph but not plasma S1P levels were significantly reduced in both groups of knockout mice. Our lipidomic results also showed that Spns2 releases different S1P species into lymph. Interestingly, lymphatic vessels in the lymph nodes (LNs) of Spns2-/- and Spns2-Mx1Cre mice exhibited morphological defects. The structures of high endothelial venules (HEV) in the LNs of Spns2-Mx1Cre mice were disorganized. These results indicate that lack of Spns2 affects both S1P secretion and LN vasculatures. Nevertheless, blood vasculature of these Spns2 deficient mice was not different to controls under homeostasis and vascular insults. Importantly, Spns2-Mx1Cre mice were resistant to multiple sclerosis in experimental autoimmune encephalomyelitis (EAE) models with significant reduction of pathogenic Th17 cells in the central nervous system (CNS). This study suggests that pharmacological inhibition of Spns2 may be exploited for therapeutic applications in treatment of neuroinflammation.

Mfsd2b and Spns2 are essential for maintenance of blood vessels during development and in anaphylactic shock.

Sphingosine-1-phosphate (S1P) is a potent lipid mediator that is secreted by several cell types. We recently showed that Mfsd2b is an S1P transporter from hematopoietic cells that contributes approximately 50% plasma S1P. Here we report the characterization of compound deletion of Mfsd2b and Spns2, another S1P transporter active primarily in endothelial cells. Global deletion of Mfsd2b and Spns2 (global double knockout [gDKO]) results in embryonic lethality beyond embryonic day 14.5 (E14.5), with severe hemorrhage accompanied by defects of tight junction proteins, indicating that Mfsd2b and Spns2 provide S1P for signaling, which is essential for blood vessel integrity. Compound postnatal deletion of Mfsd2b and Spns2 using Mx1Cre (ctDKO-Mx1Cre) results in maximal 80% reduction of plasma S1P. ctDKO-Mx1Cre mice exhibit severe susceptibility to anaphylaxis, indicating that S1P from Mfsd2b and Spns2 is indispensable for vascular homeostasis. Our results show that S1P export from Mfsd2b and Spns2 is essential for developing and mature vasculature.

A genomic biomarker that identifies human bone marrow-derived mesenchymal stem cells with high scalability.

Although the application of human mesenchymal stem cells (hMSCs) to repair damaged or diseased tissues has proven relatively effective, both the donor-to-donor variability in ex vivo expansion rates and the maintenance of stemness remain a bottleneck to widespread translation. Previous work from this laboratory stratified donors into those yielding hMSCs with high- or low-growth capacity; global transcriptomic analysis revealed that high-growth-capacity hMSCs were characterized by a loss of the gene encoding glutathione S-transferase theta 1 (GSTT1). These GSTT1-null hMSCs demonstrated increased proliferative rates, clonogenic potential, and longer telomeres compared with low-growth capacity hMSCs that were GSTT1-positive. Thus, this study identifies GSTT1 as a novel genomic DNA biomarker for hMSC scalability.

Enhancing the Efficacy of Stem Cell Therapy with Glycosaminoglycans.

Human mesenchymal stem cell (hMSC) therapy offers significant potential for osteochondral regeneration. Such applications require their ex vivo expansion in media frequently supplemented with fibroblast growth factor 2 (FGF2). Particular heparan sulfate (HS) fractions stabilize FGF2-FGF receptor complexes. We show that an FGF2-binding HS variant (HS8) accelerates the expansion of freshly isolated bone marrow hMSCs without compromising their naivety. Importantly, the repair of osteochondral defects in both rats and pigs is improved after treatment with HS8-supplemented hMSCs (MSCHS8), when assessed histologically, biomechanically, or by MRI. Thus, supplementing hMSC culture media with an HS variant that targets endogenously produced FGF2 allows the elimination of exogenous growth factors that may adversely affect their therapeutic potency.

Retention of the Structure and Function of Heparan Sulfate Biomaterials After Gamma Irradiation.

Heparan sulfate (HS) is a highly heterogeneous polysaccharide implicated in many important biological processes. Our previous work has demonstrated that a particular affinity-selected HS (referred to henceforth as "HS3") is capable of enhancing the osteogenic effects of bone morphogenetic protein 2 (BMP2). Here, we gamma-irradiated HS with 26 kGy of ionizing radiation to determine how this affected the structure, composition, and function. Initial structural studies were performed on a commercial preparation of HS as a proof-of-concept. Gamma irradiation of this HS preparation did not significantly alter its structure or composition compared to nonirradiated material, as demonstrated by proton nuclear magnetic resonance spectroscopy, molecular weight analysis using size exclusion chromatography, and disaccharide compositional analysis. When HS3 was gamma irradiated, no significant effect on binding affinity toward BMP2 was observed, based on competitive surface plasmon resonance and differential scanning fluorimetry assays. Furthermore, irradiation did not significantly affect HS3's ability to synergistically enhance the osteogenic effects of BMP2 in vitro; as measured by the relative abundance of osteogenic transcripts in transdifferentiating C2C12 murine myoblasts. Additionally, no significant differences were observed in the levels of alkaline phosphatase (ALP) or calcium deposition in C2C12s treated with BMP2, together with the irradiated, or nonirradiated HS3. Irradiation of HS3 incorporated into collagen type I sponges did not affect its ability to enhance BMP2-mediated ALP expression in C2C12 cells. Our data confirm that gamma irradiation is a cost-effective and viable solution for the sterilization of HS species that allows the retention of its structure and biological function. The work suggests an effective way to incorporate clinically compatible HS species into orthotic implants, scaffolds, and other medical devices for use in the treatment of a range of diseases and disorders.

Microfluidic Screening Reveals Heparan Sulfate Enhances Human Mesenchymal Stem Cell Growth by Modulating Fibroblast Growth Factor-2 Transport.

Cost-effective expansion of human mesenchymal stem/stromal cells (hMSCs) remains a key challenge for their widespread clinical deployment. Fibroblast growth factor-2 (FGF-2) is a key hMSC mitogen often supplemented to increase hMSC growth rates. However, hMSCs also produce endogenous FGF-2, which critically interacts with cell surface heparan sulfate (HS). We assessed the interplay of FGF-2 with a heparan sulfate variant (HS8) engineered to bind FGF-2 and potentiate its activity. Bone marrow-derived hMSCs were screened in perfused microbioreactor arrays (MBAs), showing that HS8 (50 µg/ml) increased hMSC proliferation and cell number after 3 days, with an effect equivalent to FGF-2 (50 ng/ml). In combination, the effects of HS8 and FGF-2 were additive. Differential cell responses, from upstream to downstream culture chambers under constant flow of media in the MBA, provided insights into modulation of FGF-2 transport by HS8. HS8 treatment induced proliferation mainly in the downstream chambers, suggesting a requirement for endogenous FGF-2 accumulation, whereas responses to FGF-2 occurred primarily in the upstream chambers. Adding HS8 along with FGF-2, however, maximized the range of FGF-2 effectiveness. Measurements of FGF-2 in static cultures then revealed that this was because HS8 caused increased endogenous FGF-2 production and liberated FGF-2 from the cell surface into the supernatant. HS8 also sustained levels of supplemented FGF-2 available over 3 days. These results suggest HS8 enhances hMSC proliferation and expansion by leveraging endogenous FGF-2 production and maximizing the effect of supplemented FGF-2. This is an exciting strategy for cost-effective expansion of hMSCs. Stem Cells Translational Medicine 2017;6:1178-1190.

Identification of Five Novel Salmonella Typhi-Specific Genes as Markers for Diagnosis of Typhoid Fever Using Single-Gene Target PCR Assays.

Salmonella Typhi (S. Typhi) causes typhoid fever which is a disease characterised by high mortality and morbidity worldwide. In order to curtail the transmission of this highly infectious disease, identification of new markers that can detect the pathogen is needed for development of sensitive and specific diagnostic tests. In this study, genomic comparison of S. Typhi with other enteric pathogens was performed, and 6 S. Typhi genes, that is, STY0201, STY0307, STY0322, STY0326, STY2020, and STY2021, were found to be specific in silico. Six PCR assays each targeting a unique gene were developed to test the specificity of these genes in vitro. The diagnostic sensitivities and specificities of each assay were determined using 39 S. Typhi, 62 non-Typhi Salmonella, and 10 non-Salmonella clinical isolates. The results showed that 5 of these genes, that is, STY0307, STY0322, STY0326, STY2020, and STY2021, demonstrated 100% sensitivity (39/39) and 100% specificity (0/72). The detection limit of the 5 PCR assays was 32 pg for STY0322, 6.4 pg for STY0326, STY2020, and STY2021, and 1.28 pg for STY0307. In conclusion, 5 PCR assays using STY0307, STY0322, STY0326, STY2020, and STY2021 were developed and found to be highly specific at single-gene target resolution for diagnosis of typhoid fever.