R-3 Repository :: Browsing by Author "Gao, Yang"

Browsing by Author "Gao, Yang"

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Amniotic Fluid-Derived Stem Cells Demonstrated Cardiogenic Potential in Indirect Co-culture with Human Cardiac Cells
(Springer, 2014) Gao, Yang; Connell, Jennifer Petsche; Wadhwa, Lalita; Ruano, Rodrigo; Jacot, Jeffrey G.
Amniotic fluid-derived stem cells (AFSC) have been shown to be broadly multipotent and non-tumorogenic. Previous studies of direct mixing of AFSC and neonatal rat ventricle myocytes indicated evidence of AFSC cardiogenesis. In this study, we examined human AFSC cardiogenic potential in indirect co-culture with human cardiac cells in conditions that eliminated the possibility of cell fusion. Human AFSC in contact with human cardiac cells showed expression of cardiac troponin T (cTnT) in immunohistochemistry, and no evidence of cell fusion were found through fluorescent in situ hybridization. When indirectly co-cultured with cardiac cells, human AFSC in contact with cardiac cells across a thin porous membrane showed a statistically significant increase in cTnT expression compared to non-contact conditions but lacked upregulation of calcium modulating proteins and did not have functional or morphological characteristics of mature cardiomyocytes. This suggests that contact is a necessary but not sufficient condition for AFSC cardiac differentiation in co-culture with cardiac cells.
Characterizing the Host-Pathogen Interactions and Inter-Strain Competition in Pathogenic Bacteria
(2023-06-01) Zhang, Liyang; Kirienko, Natasha V; Warmflash, Aryeh; Gao, Yang; van der Hoeven, Ransome V; Igoshin, Oleg A
Pathogens are of significant importance in medicine and public health, as they are responsible for a wide range of infectious diseases that can have serious consequences for human and animal populations. Whether infection gets established, depends on multiple factors, including environment, the host, and interactions with other bacteria. Understanding these factors and their impact is a prerequisite for developing therapies for resisting or disarming pathogenic bacteria. Adhesins are proteins present on the microbial cell surface that mediate the interactions with or attachment to the host or substance. As one type of virulence factors, adhesin proteins play a crucial role in the ability of the fungal pathogen Candida albicans to undergo cellular morphogenesis, develop robust biofilms, colonize, and cause infection in a host. By performing a comprehensive, high-throughput screen of a library of adhesin mutants in the model nematode Caenorhabditis elegans as a simplified host system, I identified mutants critical for virulence of C. albicans. Colonization is generally considered a prerequisite for infection, but this event is context-dependent, as evidenced by the differing ability of the human pathogen Pseudomonas aeruginosa to efficiently colonize C. elegans on agar but not in liquid pathogenesis. I showed that the transition to a liquid environment reduces food uptake, decreases specific adhesins, slightly upregulates host immunity, and induces a pathogen-driven dormancy of C. elegans, which restricts pathogenic colonization. My study also found that pathogenic colonization was still required for the virulence of Enterococcus faecalis even in the liquid. I conclude that poor colonization in liquid is likely due to a combination of environmental factors and host-pathogen interactions. These results provide new insights into mechanisms for colonization in different models, enabling pathogenesis models to be fine-tuned to more accurately represent the conditions seen in human infections so that new tools for curbing bacterial and fungal infections can be developed. Competition shapes the life spectrum in nature, resulting in organisms with better fitness taking a position of dominance and prevalence. The high-risk clone of P. aeruginosa ST111 predominates in hematopoietic cell transplant and hematologic malignancy (HCT/HM) bloodstream infection (BSI) patients via a fitness benefit due to the loss of functional OprD, a porin responsible for the import of carbapenems. Further study revealed that not only ST111 but also several international high-risk sequence types produce the bactericidal R5 pyocin that targets P. aeruginosa with mutations on WaaL, an O-antigen ligase of lipopolysaccharide. These findings suggest a novel approach for evaluating risks associated with emerging prevalent P. aeruginosa strains and may inform the development of strategies to mitigate the impact of ST111 and other high-risk clones on public health. In conclusion, my dissertation research provides valuable insights into the virulence of pathogens and their interactions with the host. Having a thorough understanding of the features of each infection model can power researchers to do pathogenesis research and hereby develop effective treatments or interventions for infectious diseases. The identification of R5 pyocin producers and their fitness benefits in causing infection highlights the need for ongoing monitoring and surveillance to inform public health strategies to mitigate the impact of emerging pathogens on human health.
Computationally exploring the mechanism of bacteriophage T7 gp4 helicase translocating along ssDNA
(National Academy of Sciences, 2022) Jin, Shikai; Bueno, Carlos; Lu, Wei; Wang, Qian; Chen, Mingchen; Chen, Xun; Wolynes, Peter G.; Gao, Yang; Center for Theoretical Biological Physics
Bacteriophage T7 gp4 helicase has served as a model system for understanding mechanisms of hexameric replicative helicase translocation. The mechanistic basis of how nucleoside 5′-triphosphate hydrolysis and translocation of gp4 helicase are coupled is not fully resolved. Here, we used a thermodynamically benchmarked coarse-grained protein force field, Associative memory, Water mediated, Structure and Energy Model (AWSEM), with the single-stranded DNA (ssDNA) force field 3SPN.2C to investigate gp4 translocation. We found that the adenosine 5′-triphosphate (ATP) at the subunit interface stabilizes the subunit–subunit interaction and inhibits subunit translocation. Hydrolysis of ATP to adenosine 5′-diphosphate enables the translocation of one subunit, and new ATP binding at the new subunit interface finalizes the subunit translocation. The LoopD2 and the N-terminal primase domain provide transient protein–protein and protein–DNA interactions that facilitate the large-scale subunit movement. The simulations of gp4 helicase both validate our coarse-grained protein–ssDNA force field and elucidate the molecular basis of replicative helicase translocation.
DNA Helicase–Polymerase Coupling in Bacteriophage DNA Replication
(MDPI, 2021) Lo, Chen-Yu; Gao, Yang
Bacteriophages have long been model systems to study the molecular mechanisms of DNA replication. During DNA replication, a DNA helicase and a DNA polymerase cooperatively unwind the parental DNA. By surveying recent data from three bacteriophage replication systems, we summarized the mechanistic basis of DNA replication by helicases and polymerases. Kinetic data have suggested that a polymerase or a helicase alone is a passive motor that is sensitive to the base-pairing energy of the DNA. When coupled together, the helicase–polymerase complex is able to unwind DNA actively. In bacteriophage T7, helicase and polymerase reside right at the replication fork where the parental DNA is separated into two daughter strands. The two motors pull the two daughter strands to opposite directions, while the polymerase provides a separation pin to split the fork. Although independently evolved and containing different replisome components, bacteriophage T4 replisome shares mechanistic features of Hel–Pol coupling that are similar to T7. Interestingly, in bacteriophages with a limited size of genome like Φ29, DNA polymerase itself can form a tunnel-like structure, which encircles the DNA template strand and facilitates strand displacement synthesis in the absence of a helicase. Studies on bacteriophage replication provide implications for the more complicated replication systems in bacteria, archaeal, and eukaryotic systems, as well as the RNA genome replication in RNA viruses.
DNA Polymerase-Parental DNA Interaction Is Essential for Helicase-Polymerase Coupling during Bacteriophage T7 DNA Replication
(MDPI, 2022) Lo, Chen-Yu; Gao, Yang
DNA helicase and polymerase work cooperatively at the replication fork to perform leading-strand DNA synthesis. It was believed that the helicase migrates to the forefront of the replication fork where it unwinds the duplex to provide templates for DNA polymerases. However, the molecular basis of the helicase-polymerase coupling is not fully understood. The recently elucidated T7 replisome structure suggests that the helicase and polymerase sandwich parental DNA and each enzyme pulls a daughter strand in opposite directions. Interestingly, the T7 polymerase, but not the helicase, carries the parental DNA with a positively charged cleft and stacks at the fork opening using a β-hairpin loop. Here, we created and characterized T7 polymerases each with a perturbed β-hairpin loop and positively charged cleft. Mutations on both structural elements significantly reduced the strand-displacement synthesis by T7 polymerase but had only a minor effect on DNA synthesis performed against a linear DNA substrate. Moreover, the aforementioned mutations eliminated synergistic helicase-polymerase binding and unwinding at the DNA fork and processive fork progressions. Thus, our data suggested that T7 polymerase plays a dominant role in helicase-polymerase coupling and replisome progression.
Early Drug Discovery and Development of Novel Cancer Therapeutics Targeting DNA Polymerase Eta (POLH)
(Frontiers Media S.A., 2021) Wilson, David M.; Duncton, Matthew A.J.; Chang, Caleb; Lee Luo, Christie; Georgiadis, Taxiarchis M.; Pellicena, Patricia; Deacon, Ashley M.; Gao, Yang; Das, Debanu
Polymerase eta (or Pol η or POLH) is a specialized DNA polymerase that is able to bypass certain blocking lesions, such as those generated by ultraviolet radiation (UVR) or cisplatin, and is deployed to replication foci for translesion synthesis as part of the DNA damage response (DDR). Inherited defects in the gene encoding POLH (a.k.a., XPV) are associated with the rare, sun-sensitive, cancer-prone disorder, xeroderma pigmentosum, owing to the enzyme’s ability to accurately bypass UVR-induced thymine dimers. In standard-of-care cancer therapies involving platinum-based clinical agents, e.g., cisplatin or oxaliplatin, POLH can bypass platinum-DNA adducts, negating benefits of the treatment and enabling drug resistance. POLH inhibition can sensitize cells to platinum-based chemotherapies, and the polymerase has also been implicated in resistance to nucleoside analogs, such as gemcitabine. POLH overexpression has been linked to the development of chemoresistance in several cancers, including lung, ovarian, and bladder. Co-inhibition of POLH and the ATR serine/threonine kinase, another DDR protein, causes synthetic lethality in a range of cancers, reinforcing that POLH is an emerging target for the development of novel oncology therapeutics. Using a fragment-based drug discovery approach in combination with an optimized crystallization screen, we have solved the first X-ray crystal structures of small novel drug-like compounds, i.e., fragments, bound to POLH, as starting points for the design of POLH inhibitors. The intrinsic molecular resolution afforded by the method can be quickly exploited in fragment growth and elaboration as well as analog scoping and scaffold hopping using medicinal and computational chemistry to advance hits to lead. An initial small round of medicinal chemistry has resulted in inhibitors with a range of functional activity in an in vitro biochemical assay, leading to the rapid identification of an inhibitor to advance to subsequent rounds of chemistry to generate a lead compound. Importantly, our chemical matter is different from the traditional nucleoside analog-based approaches for targeting DNA polymerases.
In crystallo observation of three metal ion promoted DNA polymerase misincorporation
(Springer Nature, 2022) Chang, Caleb; Lee Luo, Christie; Gao, Yang
Error-free replication of DNA is essential for life. Despite the proofreading capability of several polymerases, intrinsic polymerase fidelity is in general much higher than what base-pairing energies can provide. Although researchers have investigated this long-standing question with kinetics, structural determination, and computational simulations, the structural factors that dictate polymerase fidelity are not fully resolved. Time-resolved crystallography has elucidated correct nucleotide incorporation and established a three-metal-ion-dependent catalytic mechanism for polymerases. Using X-ray time-resolved crystallography, we visualize the complete DNA misincorporation process catalyzed by DNA polymerase η. The resulting molecular snapshots suggest primer 3´-OH alignment mediated by A-site metal ion binding is the key step in substrate discrimination. Moreover, we observe that C-site metal ion binding preceded the nucleotidyl transfer reaction and demonstrate that the C-site metal ion is strictly required for misincorporation. Our results highlight the essential but separate roles of the three metal ions in DNA synthesis.
Observing one-divalent-metal-ion-dependent and histidine-promoted His-Me family I-PpoI nuclease catalysis in crystallo
(eLife Sciences Publications Ltd, 2024) Chang, Caleb; Zhou, Grace; Gao, Yang
Metal-ion-dependent nucleases play crucial roles in cellular defense and biotechnological applications. Time-resolved crystallography has resolved catalytic details of metal-ion-dependent DNA hydrolysis and synthesis, uncovering the essential roles of multiple metal ions during catalysis. The histidine-metal (His-Me) superfamily nucleases are renowned for binding one divalent metal ion and requiring a conserved histidine to promote catalysis. Many His-Me family nucleases, including homing endonucleases and Cas9 nuclease, have been adapted for biotechnological and biomedical applications. However, it remains unclear how the single metal ion in His-Me nucleases, together with the histidine, promotes water deprotonation, nucleophilic attack, and phosphodiester bond breakage. By observing DNA hydrolysis in crystallo with His-Me I-PpoI nuclease as a model system, we proved that only one divalent metal ion is required during its catalysis. Moreover, we uncovered several possible deprotonation pathways for the nucleophilic water. Interestingly, binding of the single metal ion and water deprotonation are concerted during catalysis. Our results reveal catalytic details of His-Me nucleases, which is distinct from multi-metal-ion-dependent DNA polymerases and nucleases.
Primer terminal ribonucleotide alters the active site dynamics of DNA polymerase η and reduces DNA synthesis fidelity
(Elsevier, 2023) Chang, Caleb; Lee Luo, Christie; Eleraky, Sarah; Lin, Aaron; Zhou, Grace; Gao, Yang
DNA polymerases catalyze DNA synthesis with high efficiency, which is essential for all life. Extensive kinetic and structural efforts have been executed in exploring mechanisms of DNA polymerases, surrounding their kinetic pathway, catalytic mechanisms, and factors that dictate polymerase fidelity. Recent time-resolved crystallography studies on DNA polymerase η (Pol η) and β have revealed essential transient events during the DNA synthesis reaction, such as mechanisms of primer deprotonation, separated roles of the three metal ions, and conformational changes that disfavor incorporation of the incorrect substrate. DNA-embedded ribonucleotides (rNs) are the most common lesion on DNA and a major threat to genome integrity. While kinetics of rN incorporation has been explored and structural studies have revealed that DNA polymerases have a steric gate that destabilizes ribonucleotide triphosphate binding, the mechanism of extension upon rN addition remains poorly characterized. Using steady-state kinetics, static and time-resolved X-ray crystallography with Pol η as a model system, we showed that the extra hydroxyl group on the primer terminus does alter the dynamics of the polymerase active site as well as the catalysis and fidelity of DNA synthesis. During rN extension, Pol η error incorporation efficiency increases significantly across different sequence contexts. Finally, our systematic structural studies suggest that the rN at the primer end improves primer alignment and reduces barriers in C2′-endo to C3′-endo sugar conformational change. Overall, our work provides further mechanistic insights into the effects of rN incorporation on DNA synthesis.
Stem Cells and Progenitor Cells for Tissue-Engineered Solutions to Congenital Heart Defects
(Libertas Academica Ltd, 2015) Gao, Yang; Jacot, Jeffrey G.
Synthetic patches and fixed grafts currently used in the repair of congenital heart defects are nonliving, noncontractile, and not electrically responsive, leading to increased risk of complication, reoperation, and sudden cardiac death. Studies suggest that tissue-engineered patches made from living, functional cells could grow with the patient, facilitate healing, and help recover cardiac function. In this paper, we review the research into possible sources of cardiomyocytes and other cardiac cells, including embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, adipose-derived stem cells, umbilical cord blood cells, amniotic fluid-derived stem cells, and cardiac progenitor cells. Each cell source has advantages, but also has technical hurdles to overcome, including heterogeneity, functional maturity, immunogenicity, and pathogenicity. Additionally, biomaterials used as patch materials will need to attract and support desired cells and induce minimal immune responses.
Structural and dynamic basis of DNA capture and translocation by mitochondrial Twinkle helicase
(Oxford University Press, 2022) Li, Zhuo; Kaur, Parminder; Lo, Chen-Yu; Chopra, Neil; Smith, Jamie; Wang, Hong; Gao, Yang
Twinkle is a mitochondrial replicative helicase which can self-load onto and unwind mitochondrial DNA. Nearly 60 mutations on Twinkle have been linked to human mitochondrial diseases. Using cryo-electron microscopy (cryo-EM) and high-speed atomic force microscopy (HS-AFM), we obtained the atomic-resolution structure of a vertebrate Twinkle homolog with DNA and captured in real-time how Twinkle is self-loaded onto DNA. Our data highlight the important role of the non-catalytic N-terminal domain of Twinkle. The N-terminal domain directly contacts the C-terminal helicase domain, and the contact interface is a hotspot for disease-related mutations. Mutations at the interface destabilize Twinkle hexamer and reduce helicase activity. With HS-AFM, we observed that a highly dynamic Twinkle domain, which is likely to be the N-terminal domain, can protrude ∼5 nm to transiently capture nearby DNA and initialize Twinkle loading onto DNA. Moreover, structural analysis and subunit doping experiments suggest that Twinkle hydrolyzes ATP stochastically, which is distinct from related helicases from bacteriophages.
Structural basis for the activation of a compact CRISPR-Cas13 nuclease
(Springer Nature, 2023) Deng, Xiangyu; Osikpa, Emmanuel; Yang, Jie; Oladeji, Seye J.; Smith, Jamie; Gao, Xue; Gao, Yang
The CRISPR-Cas13 ribonucleases have been widely applied for RNA knockdown and transcriptional modulation owing to their high programmability and specificity. However, the large size of Cas13 effectors and their non-specific RNA cleavage upon target activation limit the adeno-associated virus based delivery of Cas13 systems for therapeutic applications. Herein, we report detailed biochemical and structural characterizations of a compact Cas13 (Cas13bt3) suitable for adeno-associated virus delivery. Distinct from many other Cas13 systems, Cas13bt3 cleaves the target and other nonspecific RNA at internal “UC” sites and is activated in a target length-dependent manner. The cryo-electron microscope structure of Cas13bt3 in a fully active state illustrates the structural basis of Cas13bt3 activation. Guided by the structure, we obtain engineered Cas13bt3 variants with minimal off-target cleavage yet maintained target cleavage activities. In conclusion, our biochemical and structural data illustrate a distinct mechanism for Cas13bt3 activation and guide the engineering of Cas13bt3 applications.
Structural basis of the stereoselective formation of the spirooxindole ring in the biosynthesis of citrinadins
(Springer Nature, 2021) Liu, Zhiwen; Zhao, Fanglong; Zhao, Boyang; Yang, Jie; Ferrara, Joseph; Sankaran, Banumathi; Venkataram Prasad, B.V.; Kundu, Biki Bapi; Phillips, George N.Jr.; Gao, Yang; Hu, Liya; Zhu, Tong; Gao, Xue
Prenylated indole alkaloids featuring spirooxindole rings possess a 3R or 3S carbon stereocenter, which determines the bioactivities of these compounds. Despite the stereoselective advantages of spirooxindole biosynthesis compared with those of organic synthesis, the biocatalytic mechanism for controlling the 3R or 3S-spirooxindole formation has been elusive. Here, we report an oxygenase/semipinacolase CtdE that specifies the 3S-spirooxindole construction in the biosynthesis of 21R-citrinadin A. High-resolution X-ray crystal structures of CtdE with the substrate and cofactor, together with site-directed mutagenesis and computational studies, illustrate the catalytic mechanisms for the possible β-face epoxidation followed by a regioselective collapse of the epoxide intermediate, which triggers semipinacol rearrangement to form the 3S-spirooxindole. Comparing CtdE with PhqK, which catalyzes the formation of the 3R-spirooxindole, we reveal an evolutionary branch of CtdE in specific 3S spirocyclization. Our study provides deeper insights into the stereoselective catalytic machinery, which is important for the biocatalysis design to synthesize spirooxindole pharmaceuticals.
Sugar ring alignment and dynamics underline cytarabine and gemcitabine inhibition on Pol η catalyzed DNA synthesis
(Elsevier, 2024) Chang, Caleb; Zhou, Grace; Lee Luo, Christie; Eleraky, Sarah; Moradi, Madeline; Gao, Yang
Nucleoside analogue drugs are pervasively used as antiviral and chemotherapy agents. Cytarabine and gemcitabine are anti-cancer nucleoside analogue drugs that contain C2′ modifications on the sugar ring. Despite carrying all the required functional groups for DNA synthesis, these two compounds inhibit DNA extension once incorporated into DNA. It remains unclear how the C2′ modifications on cytarabine and gemcitabine affect the polymerase active site during substrate binding and DNA extension. Using steady-state kinetics, static and time-resolved X-ray crystallography with DNA polymerase η (Pol η) as a model system, we showed that the sugar ring C2′ chemical groups on cytarabine and gemcitabine snugly fit within the Pol η active site without occluding the steric gate. During DNA extension, Pol η can extend past gemcitabine but with much lower efficiency past cytarabine. The Pol η crystal structures show that the -OH modification in the β direction on cytarabine locks the sugar ring in an unfavorable C2′-endo geometry for product formation. On the other hand, the addition of fluorine atoms on gemcitabine alters the proper conformational transition of the sugar ring for DNA synthesis. Our study illustrates mechanistic insights into chemotherapeutic drug inhibition and resistance and guides future optimization of nucleoside analogue drugs.
Understanding the Mechanisms of DNA Polymerases and Nucleases with Time-Resolved X-ray Crystallography
(2024-04-18) Chang, Caleb; Gao, Yang
Divalent metal ions, especially Mg2+, play pivotal roles in an enzyme’s ability to manipulate the highly stable structure of DNA. DNA and RNA polymerases, as well as numerous nucleases are clear examples of such enzymes, and are integral to critical cellular functions. Thus, these proteins represent important drug targets for various diseases and biotechnological tools for genome editing. Understanding the molecular mechanism of Mg2+-promoted DNA synthesis and cleavage is crucial for engineering and efficiently targeting of these enzymes. Time-resolved X-ray crystallography enables the visualization of catalytic processes and aids in dissecting catalytic molecular mechanisms. This technique tracks active conformations and intermediate states during catalysis by initiating chemical reactions in protein crystals synchronously via light activation or substrate diffusion. In my thesis work, I applied diffusion-based time-resolved crystallography to investigate two representative enzymes: DNA polymerase η, which is a Y-family DNA polymerase that participates during DNA replication to bypass bulky DNA lesions; and I-PpoI endonuclease, which is one-metal dependent His-Me nuclease that has an active site homologous to the HNH active site of the Cas9 nuclease. In elucidating the catalytic mechanism of DNA polymerases, wild-type and mutant variants of DNA polymerase η were generated and analyzed using kinetic assays. Over 100 crystal structures of DNA polymerase η complexed with a wide variety of deoxyribonucleotides, ribonucleotides, and nucleoside analogue drugs were determined with resolutions ranging from 1.4 to 2.8 Å, providing high-resolution visualization of canonical DNA synthesis and polymerase targeting. The structural alignments revealed the essential role of the third divalent metal ion and the dynamics of the primer end, including the sugar ring, correlated to substrate discrimination and efficient chemistry. Similarly, I tracked the reaction process of I-PpoI with kinetic assays and time-resolved crystallography. More than 40 crystal structures of I-PpoI at various pH and metal ion concentrations were determined. The intermediate structures revealed the involvement of one and only one divalent metal ion in DNA hydrolysis. DNA cleavage assays unveiled several possible deprotonation pathways for the nucleophilic water molecule. Importantly, metal ion binding and water deprotonation were found to be highly correlated during catalysis. These results offer mechanistic insights that can be instrumental in enhancing the bioengineering and targeting of polymerases and nucleases.
Use of Human Pediatric Cardiac Progenitor Cells in an Engineered Heart Patch
(2015-09-30) Gao, Yang; Jacot, Jeffrey G; Grande-Allen, Jane; Harrington, Daniel
Congenital heart defects (CHD) are the most common birth defects in the US and the leading cause of death in newborns. Some of the most prevalent CHD require surgical interventions with patch materials. However, current commercial patch materials are acellular, non-conductive, and non-contractile; they can induce arrhythmias and require reoperations. We envision an engineered cardiac patch seeded with autologous cells from the patient. However, mature cardiomyocytes (CM) rarely proliferate. This research examined the ability of primary pediatric cardiac cells (PPCC) isolated from pediatric CHD biopsy samples supplied by Texas Children’s Hospital to differentiate into CM or induce CM differentiation in stem cells. Previous studies indicated evidence of cardiogenesis in Amniotic fluid-derived stem cells (AFSC) when directly mixed with neonatal rat ventricle myocytes. We hypothesized that co-culturing with human PPCC will induce cardiac differentiation in human AFSC (hAFSC). hAFSC co-cultured in contact with PPCC showed a statistically significant increase in cTnT expression compared to non-contact conditions but did not have functional or morphological characteristics of mature cardiomyocytes. This result suggests that contact is a necessary but not sufficient condition for AFSC cardiac differentiation in co-culture with PPCC. Cardiac progenitor cells (CPC) are proliferating cells with the ability to differentiate into cardiac cells. CPC can be identified from cardiac cells by the expression of Isl-1, SSEAs, and c-Kit. We hypothesized that there are potential CPC in PPCC. We found a small subpopulation (1%-4%) of the primary cells expressing Isl-1, SSEA-4, and c-Kit. However, when exposed to oxytocin, PPCC did not differentiate into functional CM as shown with murine CPC in literature. Extracellular matrix proteins isolated from adult cardiac tissue have been shown to promote CM maturation in vitro. PPCC can be expanded in vitro and cultured in PEGylated fibrin hydrogels. PPCC conditioned gels can then be decellularized. We found that PPCC could be cultured in fibrin hydrogels and that stem cell derived CM were viable when cultured on these conditioned gels. Overall, this research demonstrated that PPCC are a potential tool for CM differentiation and maturation in the development of a tissue engineered cardiac patch for repair of CHD.

Browsing by Author "Gao, Yang"

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