Rajneesh Jha

Microscale Generation of Cardiospheres Promotes Robust Enrichment of Cardiomyocytes Derived from Human Pluripotent Stem Cells

Summary Cardiomyocytes derived from human pluripotent stem cells (hPSCs) are a promising cell source for regenerative medicine, disease modeling, and drug discovery, all of which require enriched cardiomyocytes, ideally ones with mature phenotypes. However, current methods are typically performed in 2D environments that produce immature cardiomyocytes within heterogeneous populations. Here, we generated 3D aggregates of cardiomyocytes (cardiospheres) from 2D differentiation cultures of hPSCs using microscale technology and rotary orbital suspension culture. Nearly 100% of the cardiospheres showed spontaneous contractility and synchronous intracellular calcium transients. Strikingly, from starting heterogeneous populations containing ∼10%–40% cardiomyocytes, the cell population within the generated cardiospheres featured ∼80%–100% cardiomyocytes, corresponding to an enrichment factor of up to 7-fold. Furthermore, cardiomyocytes from cardiospheres exhibited enhanced structural maturation in comparison with those from a parallel 2D culture. Thus, generation of cardiospheres represents a simple and robust method for enrichment of cardiomyocytes in microtissues that have the potential use in regenerative medicine as well as other applications.

Molecular beacon-based detection and isolation of working-type cardiomyocytes derived from human pluripotent stem cells

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) provide a potential source of cells to repair injured ventricular myocardium. CM differentiation cultures contain non-cardiac cells and CMs of both nodal and working subtypes. Direct application of such cultures in clinical studies could induce arrhythmias; thus, further purification of working-type CMs from heterogeneous cultures is desirable. Here, we designed 10 molecular beacons (MBs) targeting NPPA mRNA, a marker associated with working-type CMs and highly up-regulated during differentiation. We examined these MBs by solution assays and established their specificity using NPPA-overexpressing CHO cells as well as hPSC-CMs. We selected one MB for subsequent CM subtype isolation using fluorescence-activated cell sorting because the signal-to-background ratio was the highest for this MB in solution assays and a linear correlation was observed between MB signals and the CM purity in differentiation cultures. Compared with cells with low MB signals, cells positively selected based on MB signal had higher expression levels of genes associated with working-type CMs and lower expression levels of genes associated with nodal-type CMs. Therefore, the MB-based method is capable of separating working-type CMs from nodal-type CMs with high specificity and throughput, potentially providing working-type CMs for biomedical applications.

Chlamydia trachomatis Alters Iron-Regulatory Protein-1 Binding Capacity and Modulates Cellular Iron Homeostasis in HeLa-229 Cells

Chlamydia trachomatis (CT) is the leading cause of diseases related to reproductive health and iron plays important role in chlamydial pathogenesis. Iron homeostasis in chlamydia-infected cells is not clear thus far. This study shows that expression of the transferrin receptor (TfR) is downregulated, whereas expression of the ferritin heavy chain is upregulated in CT-infected HeLa-229 cells. Expression of iron-regulatory protein (IRP)-1 predominates over IRP-2 in infected cells. In infected cells, attenuated binding activity of IRP-iron responsive elements (IREs) is observed using the electrophoretic mobility-shift assay. These results suggest that iron homeostasis is modulated in CT-infected HeLa cells at the interface of acquisition and commensal use of iron.

Simulated Microgravity and 3D Culture Enhance Induction, Viability, Proliferation and Differentiation of Cardiac Progenitors from Human Pluripotent Stem Cells.

Efficient generation of cardiomyocytes from human pluripotent stem cells is critical for their regenerative applications. Microgravity and 3D culture can profoundly modulate cell proliferation and survival. Here, we engineered microscale progenitor cardiac spheres from human pluripotent stem cells and exposed the spheres to simulated microgravity using a random positioning machine for 3 days during their differentiation to cardiomyocytes. This process resulted in the production of highly enriched cardiomyocytes (99% purity) with high viability (90%) and expected functional properties, with a 1.5 to 4-fold higher yield of cardiomyocytes from each undifferentiated stem cell as compared with 3D-standard gravity culture. Increased induction, proliferation and viability of cardiac progenitors as well as up-regulation of genes associated with proliferation and survival at the early stage of differentiation were observed in the 3D culture under simulated microgravity. Therefore, a combination of 3D culture and simulated microgravity can be used to efficiently generate highly enriched cardiomyocytes.

Efficient Differentiation of Cardiomyocytes from Human Pluripotent Stem Cells with Growth Factors

Human pluripotent stem cells have tremendous replicative capacity and demonstrated potential to generate functional cardiomyocytes. These cardiomyocytes represent a promising source for cell replacement therapy to treat heart disease and may serve as a useful tool for drug discovery and disease modeling. Efficient cardiomyocyte differentiation, a prerequisite for the application of stem cell-derived cardiomyocytes, can be achieved with a growth factor-guided method. Undifferentiated cells are sequentially treated with activin A and BMP4 in a serum-free and insulin-free medium and then maintained in a serum-free medium with insulin. This method yields as much as >75% cardiomyocytes in the differentiation culture within 2 weeks, and the beating cardiomyocytes have expected molecular, cellular, and electrophysiological characteristics. In this chapter, we describe in detail the differentiation protocol and follow-up characterization focusing on immunocytochemistry, quantitative RT-PCR, and flow cytometry analysis.

Differing Effects of Azithromycin and Doxycycline on Cytokines in Cells from Chlamydia trachomatis–Infected Women

Chlamydial infection of the lower genital tract usually spreads to the upper genital tract and is then responsible for more serious consequences, such as infertility, ectopic pregnancy, pelvic pain, and pelvic inflammatory disease. Genital infection with Chlamydia trachomatis and the resulting cytokine response largely determines the outcome of infection and disease. To date, studies showing comparative effects of azithromycin and doxycycline treatment for C. trachomatis infection in women with reproductive sequelae like infertility and their effect on immune molecules like cytokines are lacking. Hence, our objective was to study the effect of azithromycin and doxycycline in vitro on cytokines in cells from C. trachomatis-positive fertile and infertile women as well as their efficacy in C. trachomatis infection. Fertile and infertile women with primary and recurrent C. trachomatis infection attending the gynecology outpatient department of Safdarjung Hospital, New Delhi, India, were enrolled. Enzyme-linked immunosorbent assay and real-time reverse transcription-polymerase chain reaction was performed for evaluating cytokines in cells stimulated with chlamydial elementary bodies (EBs) in the presence and absence of antibiotics (azithromycin and doxycycline). C. trachomatis-infected women were also followed up to assess the efficacy of azithromycin and doxycycline. We observed inhibition of cytokines (interleukin [IL]-1beta (β), IL-6, IL-8, IL-10, and tumor necrosis factor-alpha) in the presence of azithromycin in EB-stimulated cells from both fertile and infertile women with primary and recurrent C. trachomatis infection. However, in presence of doxycycline, inhibition of cytokines (IL-1β and IL-6) was only observed in stimulated cells from fertile women with primary C. trachomatis infection. The clinical efficacy of azithromycin was also better than doxycycline in recurrent C. trachomatis infection in women with complications such as infertility. Overall, this study suggests that azithromycin treatment with broader immunomodulatory effects may be preferable to doxycycline for the treatment of recurrent C. trachomatis infection associated with infertility.

Azithromycin Treatment Modulates the Extracellular Signal-Regulated Kinase Mediated Pathway and Inhibits Inflammatory Cytokines and Chemokines in Epithelial Cells from Infertile Women with Recurrent Chlamydia trachomatis Infection

Epidemiological and animal model studies suggest that sequelae of genital Chlamydia trachomatis infection are more often associated with second or subsequent infections than with initial infection. Further, in order to establish an acute or long-term persistent infection, C. trachomatis develops several strategies to circumvent host immune responses. Hence, resolution of the C. trachomatis infection may require modulation of host factors especially during persistent or chronic infection. Moreover, azithromycin treatment has been reported to possess anti-inflammatory properties but its mechanism of action is still not elucidated. Therefore, in order to better understand the effect of azithromycin in chronic conditions, our aim was to study changes in expression of key genes associated with inflammatory cytokines and receptors, mitogen-activated protein kinase (MAPK) signaling pathway, and apoptosis pathway before and after therapy with azithromycin in infertile women with recurrent C. trachomatis infection. Real-time polymerase chain reaction was performed to study inflammatory cytokines and receptors, MAPK signaling pathway, and apoptosis pathway before and after therapy with azithromycin in infertile women with recurrent C. trachomatis infection. Further, effect of azithromycin on activation of extracellular signal-regulated kinase was studied in epithelial cells by western blotting. Chemokine (C-C motif) ligand 2 (CCL2), CCL5, chemokine (C-X-C motif) ligand 1 (CXCL1), CXCL5, CXCL9, interleukin-1B (IL-1B), IL-8, baculoviral IAP repeat-containing 3 (BIRC3), myeloid cell leukemia sequence 1 (MCL1), and MAPK1 were downregualted after azithromycin treatment. In addition, phosphorylation of extracellular signal-regulated kinase was inhibited after azithromycin treatment in epithelial cells obtained from women with recurrent infection. Hence, our data suggest that azithromycin with its properties apart from antibacterial activity may contribute to its therapeutic potential in treatment of chronic recurrent infection in infertile women.

Human iPSC-derived mesenchymal stem cells encapsulated in PEGDA hydrogels mature into valve interstitial-like cells

Despite recent advances in tissue engineered heart valves (TEHV), a major challenge is identifying a cell source for seeding TEHV scaffolds. Native heart valves are durable because valve interstitial cells (VICs) maintain tissue homeostasis by synthesizing and remodeling the extracellular matrix. This study demonstrates that induced pluripotent stem cells (iPSC)-derived mesenchymal stem cells (iMSCs) can be derived from iPSCs using a feeder-free protocol and then further matured into VICs by encapsulation within 3D hydrogels. The differentiation efficiency was characterized using flow cytometry, immunohistochemistry staining, and trilineage differentiation. Using our feeder-free differentiation protocol, iMSCs were differentiated from iPSCs and had CD90+, CD44+, CD71+, αSMA+, and CD45- expression. Furthermore, iMSCs underwent trilineage differentiation when cultured in induction media for 21 days. iMSCs were then encapsulated in poly(ethylene glycol)diacrylate (PEGDA) hydrogels grafted with adhesion peptide (RGDS) to promote remodeling and further maturation into VIC-like cells. VIC phenotype was assessed by the expression of alpha-smooth muscle actin (αSMA), vimentin, and collagen production after 28 days. When MSC-derived cells were encapsulated in PEGDA hydrogels that mimic the leaflet modulus, a decrease in αSMA expression and increase in vimentin was observed. In addition, iMSCs synthesized collagen type I after 28 days in 3D hydrogel culture. Thus, the results from this study suggest that iMSCs may be a promising cell source for TEHV. STATEMENT OF SIGNIFICANCE Developing a suitable cell source is a critical component for the success and durability of tissue engineered heart valves. The significance of this study is the generation of iPSCs-derived mesenchymal stem cells (iMSCs) that have the capacity to mature into valve interstitial-like cells when introduced into a 3D cell culture designed to mimic the layers of the valve leaflet. iMSCs were generated using a feeder-free protocol, which is one major advantage over other methods, as it is more clinically relevant. In addition to generating a potential new cell source for heart valve tissue engineering, this study also highlights the importance of a 3D culture environment to influence cell phenotype and function.

Targeted Elimination of Tumorigenic Human Pluripotent Stem Cells Using Suicide-inducing Virus-like Particles

Sensitization to prodrugs via transgenic expression of suicide genes is a leading strategy for the selective elimination of potentially tumorigenic human pluripotent stem cells (hPSCs) in regenerative medicine, but transgenic modification poses safety risks such as deleterious mutagenesis. We describe here an alternative method of delivering suicide-inducing molecules explicitly to hPSCs using virus-like particles (VLPs) and demonstrate its use in eliminating undifferentiated hPSCs in vitro. VLPs were engineered from Qβ bacteriophage capsids to contain enhanced green fluorescent protein (EGFP) or cytosine deaminase (CD) and to simultaneously display multiple IgG-binding ZZ domains. After labeling with antibodies against the hPSC-specific surface glycan SSEA-5, EGFP-containing particles were shown to specifically bind undifferentiated cells in culture, and CD-containing particles were able to eliminate undifferentiated hPSCs with virtually no cytotoxicity to differentiated cells upon treatment with the prodrug 5-fluorocytosine.

Downregulation of LGR5 Expression Inhibits Cardiomyocyte Differentiation and Potentiates Endothelial Differentiation from Human Pluripotent Stem Cells

Summary Understanding molecules involved in differentiation of human pluripotent stem cells (hPSCs) into cardiomyocytes and endothelial cells is important in advancing hPSCs for cell therapy and drug testing. Here, we report that LGR5, a leucine-rich repeat-containing G-protein-coupled receptor, plays a critical role in hPSC differentiation into cardiomyocytes and endothelial cells. LGR5 expression was transiently upregulated during the early stage of cardiomyocyte differentiation, and knockdown of LGR5 resulted in reduced expression of cardiomyocyte-associated markers and poor cardiac differentiation. In contrast, knockdown of LGR5 promoted differentiation of endothelial-like cells with increased expression of endothelial cell markers and appropriate functional characteristics, including the ability to form tube-like structures and to take up acetylated low-density lipoproteins. Furthermore, knockdown of LGR5 significantly reduced the proliferation of differentiated cells and increased the nuclear translocation of β-catenin and expression of Wnt signaling-related genes. Therefore, regulation of LGR5 may facilitate efficient generation of cardiomyocytes or endothelial cells from hPSCs.