2009 — 2013 |
Kwon, Chulan |
K99Activity Code Description: To support the initial phase of a Career/Research Transition award program that provides 1-2 years of mentored support for highly motivated, advanced postdoctoral research scientists. R00Activity Code Description: To support the second phase of a Career/Research Transition award program that provides 1 -3 years of independent research support (R00) contingent on securing an independent research position. Award recipients will be expected to compete successfully for independent R01 support from the NIH during the R00 research transition award period. |
Isl1 and Wntbeta-Catenin Regulation of Cardiac Progenitor Cells @ J. David Gladstone Institutes
DESCRIPTION (provided by applicant): Despite decades of progress in cardiovascular biology, heart disease remains the leading cause of death in the developed world. Recently, therapies based on cardiac progenitor cells (CPCs) have emerged as promising potential cardiac therapeutics. However, we do not know the mechanisms underlying CRC self renewal, proliferation and differentiation, a prerequisite for cardiac regenerative therapy. My recent findings suggest that canonical Wnt signaling is required for CRC expansion. To search for downstream mediators of Wnt/p-catenin signaling, I performed expression array analyses of CRCs from mouse embryos with and without constitutively active Wnt/p-catenin signaling. This search revealed several genes with pivotal roles in CRC development that are negatively affected by p-catenin, including Isietl, Myocd and Smyd1. Notably, |3- catenin stabilization severely downregulated isietl, a temporal marker of undifferentiated CRCs that is required for normal cardiogenesis. /s/ef t-deficiency increased the number of CRCs and suppressed their cardiac differentiation. To understand how cellular decisions are implemented by Isietl and Wnt/p-catenin signaling, I have begun to identify the critical downstream pathways that affect CRC maintenance and differentiation. I found that Isietl loss-of-function severely compromised expression of Myocd, a gene required for smooth muscle differentiation. Interestingly, Isietl did not affect expression of Smyd1, an essential gene for cardiomyocyte differentiation, suggesting an Isietl-independent regulation of Stnydl by Wnt/p-catenin signaling. Among the genes upregulated by p-catenin, I found that Bhlhb2, a transcriptional repressor with a novel role in cardiogenesis, is expressed specifically in the cardiac region. Importantly, increased Bhihb2 \e\/e\s in CPCs resulted in downregulation of Smyd1, raising the possibility that Wnt/pcatenin signaling may regulate Smyd1 expression through Bhlhb2. Taken together, these findings set the stage for a mechanistic exploration of the role of Wnt/p-catenin signaling and Isietl in understanding maintenance and differentiation of CRCs that will facilitate future cell-based heart therapeutics. The specific aims of this proposal are (1) To test whether Isietl affects the self-renewal, proliferation, and differentiation of CRCs;(2) To determine if Isietl is an essential effector for Wnt/p-catenin signaling-mediated expansion of CRCs;(3) To investigate if Isietl and Wnt/p-catenin signaling directly activate Myoccf and Bhlhb2, respectively, to affect CRC fates. These studies will lay the foundation for understanding the molecular mechanisms of CRC maintenance and differentiation.
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0.945 |
2012 — 2016 |
Kwon, Chulan |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Non-Canonical Notch Regulation of Cardiovascular Progenitors @ Johns Hopkins University
DESCRIPTION (provided by applicant): Cardiovascular progenitor cells (CPCs) hold tremendous therapeutic potential for cardiac regenerative medicine due to their unique ability to expand and to differentiate into various heart cell types. However, to take advantage of regenerative therapy, we need to understand the mechanisms underlying the self-renewal and lineage-specific differentiation of CPCs. The current proposal focuses on elucidating a novel role of Notch signaling in CPC maintenance and differentiation. Notch is an evolutionarily conserved transmembrane protein that plays critical roles in numerous cell-fate decisions. Canonical Notch signaling is initiated by binding of extracellular ligands to Notch. This leads to intracellular cleavage and translocation of Notch into the nucleus where it binds to the transcriptional mediator RBP-J for gene activation. I demonstrated that Notch1-deficient CPCs expand dramatically with increased proliferation, similar to the phenotype of CPCs stimulated with active 2-Catenin. This phenotype is not observed in CPCs deficient for RBP-J, suggesting a non-canonical role of Notch. Notch1-deficiency significantly increased 2-Catenin signaling. This increase was not mediated by upregulation of 2-Catenin mRNA but rather by accumulation of active 2-Catenin protein, suggesting post- translational regulation. Intriguingly, the Notch regulation of 2-Catenin protein did not require classical ligand- dependent membrane cleavage of Notch or the 2-Catenin directed proteasomal degradation, but it did require endocytic proteins that traffic membranous Notch to the lysosome. Moreover, membrane-bound Notch, conventionally considered biologically inert, physically associated with active 2-Catenin and inhibited accumulation of active 2-Catenin protein. These findings reveal a previously undescribed role of membrane Notch in regulating active 2-Catenin protein levels and set the stage for a mechanistic exploration of this role in the maintenance and differentiation of CPCs. I propose to test the hypothesis that Notch antagonizes CPC self-renewal/expansion by lysosomal degradation of active 2-Catenin in a ligand/transcription-independent fashion. The specific aims of this proposal are (1) To determine if Notch1-deficient CPCs favor self-renewal over differentiation and if 2-Catenin is required for this effect; (2) To test whether membrane-bound Notch1 affects CPC expansion and differentiation and whether the cellular events require 2-Catenin; (3) To identify the role of lysosomal activity in the link between membrane-bound Notch and active 2-Catenin degradation. The proposed work will provide fundamental insights into the understanding of mechanisms controlling CPC self- renewal/differentiation decisions, a prerequisite for CPC-mediated cardiac regenerative therapeutics. The biology of membrane Notch is completely unexplored in the field of CPCs as well as in stem cells. Given highly conserved roles of Notch and Wnt/2-Catenin signaling in nearly all known stem/progenitor cell fate decisions, these studies will open up new avenues of research for regenerative medicine involving stem/progenitor cells. PUBLIC HEALTH RELEVANCE: Understanding the biology of the cells that develop into the heart, called multipotent cardiovascular progenitor cells (CPCs), is key to realizing the promise of future cell-mediated cardiac therapeutics. The proposed research aims to elucidate the ligand/transcription-independent role of a protein called Notch in CPC self- renewal and differentiation. Our work will provide mechanistic insights into understanding the self-renewal- differentiation processes of multipotent CPCs regulated by Notch, which will facilitate future cell-based cardiac therapeutics and open new avenues of investigation for non-canonical Notch biology.
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0.945 |
2016 |
Kwon, Chulan |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulation of Cardiac Progenitor Maintenance @ Johns Hopkins University
Cardiac progenitor cells (CPCs)?identified in early embryos?are cell sources to make the heart during fetal development, and abnormal CPC development is closely associated with the etiology of congenital heart defects. In particular, proper regulation of their number and fate is essential for the ensuing heart growth. However, it remained unknown if they undergo self-renewal and if there is a dedicated environment for their maintenance. We have identified a renewing population of CPCs and their niche during development, and this proposal aims to elucidate the mechanisms governing the self-renewal and expansion of CPCs in vivo. Numb family proteins (NFPs) are conserved endocytic proteins with critical roles in cell-fate decisions. We found that precardiac deletion of NFPs depletes CPCs in the second pharyngeal arch (PA2) and is associated with a hypoplastic heart and early embryonic lethality. Based on this phenotype, we hypothesized that CPCs expand in the PA2 before their cardiac differentiation. Confirming the hypothesis, CPCs normally remained undifferentiated and expansive in the PA2, and differentiated into cardiac cells soon as they migrated out of the PA2. CPCs co-cultured with PA2 cells formed distinct colonies that continued to grow without cardiac differentiation, and differentiated into cardiac cells when PA2 cells were removed. These suggested that CPCs proliferate without differentiation in the PA2, suggesting the presence of a stem cell?niche paradigm. The proliferation was promoted by Hedgehog (Hh) proteins, crucial developmental and stem cell regulators enriched in the PA2, implying the Hh signaling may mediate the environmental role of PA2 cells. To determine the CPC-autonomous role of NFPs in the PA2, we generated lineage-specific mosaicism that allowed tracing of CPCs lacking NFPs without causing the lethality. The NFP-deleted mutant CPCs normally populated in the PA2, but failed to expand in the PA2 and progressed to cardiac cells. The mutant CPCs showed dramatically decreased levels of the conserved stem cell regulator Itgb1. Itgb1 physically associated with NFP in CPCs, and precardiac deletion of Itgb1 resulted in CPC depletion in the PA2, similar to the phenotype of NFP-deleted embryos. These findings set the stage for a mechanistic exploration of CPC maintenance by intrinsic and extrinsic factors. The specific aims of this proposal are (1) to determine if NFPs play an instructive role for CPC renewal in the PA2, (2) to determine if NFPs prevent endolysosomal degradation of Itgb1 for CPC maintenance, and (3) To investigate if PA2 cells affect CPC renewal and expansion via Hh signaling. With these aims, I expect to elucidate the factors and mechanisms by which CPCs are maintained in a renewing state in their microenvironment. This knowledge will provide first insights into the mechanistic understanding of the self-renewal of CPCs in their microenvironment during heart development, which will open up new avenues of research in congenital heart disease and may allow us to maintain and expand CPCs in a homogeneous and undifferentiated state in vitro for heart regeneration research.
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0.945 |
2017 — 2020 |
Kwon, Chulan |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulation of Cardiac Progenitor Maintece @ Johns Hopkins University
Cardiac progenitor cells (CPCs)?identified in early embryos?are cell sources to make the heart during fetal development, and abnormal CPC development is closely associated with the etiology of congenital heart defects. In particular, proper regulation of their number and fate is essential for the ensuing heart growth. However, it remained unknown if they undergo self-renewal and if there is a dedicated environment for their maintenance. We have identified a renewing population of CPCs and their niche during development, and this proposal aims to elucidate the mechanisms governing the self-renewal and expansion of CPCs in vivo. Numb family proteins (NFPs) are conserved endocytic proteins with critical roles in cell-fate decisions. We found that precardiac deletion of NFPs depletes CPCs in the second pharyngeal arch (PA2) and is associated with a hypoplastic heart and early embryonic lethality. Based on this phenotype, we hypothesized that CPCs expand in the PA2 before their cardiac differentiation. Confirming the hypothesis, CPCs normally remained undifferentiated and expansive in the PA2, and differentiated into cardiac cells soon as they migrated out of the PA2. CPCs co-cultured with PA2 cells formed distinct colonies that continued to grow without cardiac differentiation, and differentiated into cardiac cells when PA2 cells were removed. These suggested that CPCs proliferate without differentiation in the PA2, suggesting the presence of a stem cell?niche paradigm. The proliferation was promoted by Hedgehog (Hh) proteins, crucial developmental and stem cell regulators enriched in the PA2, implying the Hh signaling may mediate the environmental role of PA2 cells. To determine the CPC-autonomous role of NFPs in the PA2, we generated lineage-specific mosaicism that allowed tracing of CPCs lacking NFPs without causing the lethality. The NFP-deleted mutant CPCs normally populated in the PA2, but failed to expand in the PA2 and progressed to cardiac cells. The mutant CPCs showed dramatically decreased levels of the conserved stem cell regulator Itgb1. Itgb1 physically associated with NFP in CPCs, and precardiac deletion of Itgb1 resulted in CPC depletion in the PA2, similar to the phenotype of NFP-deleted embryos. These findings set the stage for a mechanistic exploration of CPC maintenance by intrinsic and extrinsic factors. The specific aims of this proposal are (1) to determine if NFPs play an instructive role for CPC renewal in the PA2, (2) to determine if NFPs prevent endolysosomal degradation of Itgb1 for CPC maintenance, and (3) To investigate if PA2 cells affect CPC renewal and expansion via Hh signaling. With these aims, I expect to elucidate the factors and mechanisms by which CPCs are maintained in a renewing state in their microenvironment. This knowledge will provide first insights into the mechanistic understanding of the self-renewal of CPCs in their microenvironment during heart development, which will open up new avenues of research in congenital heart disease and may allow us to maintain and expand CPCs in a homogeneous and undifferentiated state in vitro for heart regeneration research.
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0.945 |
2021 |
Kim, Deok-Ho Kwon, Chulan |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Transcriptomic Entropy to Quantify Maturation of Psc-Derived Cardiomyocytes @ Johns Hopkins University
PROJECT SUMMARY The immaturity of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) has emerged as a major challenge for their broad applicability. For this reason, extensive biological and engineering efforts are underway with the goal to generate mature, adult-like CMs from PSCs. However, owing to the lack of quantitative metrics to benchmark the maturation status of PSC-CMs, the efforts are being made largely on an ad hoc basis, and this leaves the degree and direction of PSC-CM maturation unclear. To address this, we have developed a quantitative metric based on an entropy concept that enables cross-comparison studies robust to experimental variability and species difference. With this finding, this proposal aims to apply entropy score to determine the status and trajectory of PSC-CM maturation achieved by molecular stimulation, tissue engineering, and in vivo transplantation, combined with functional and structural analysis. The use of entropy score is expected to reveal strengths and weaknesses of the ongoing approaches and inform us potential causes of maturation arrest and deviations. Thus, the proposed research will provide mechanistic insights into instructing PSC-CM to become adult CMs as well as quantitative insights into the progress made towards PSC-CM maturation in the field.
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0.945 |