Ruchi Bajpai

PIBBS MENTOR

Assistant Professor
CCMB and Biochemistry

Research Topics

Epigenetic regulation of craniofacial development using pluripotent stem cells and early embryos as model organisms

Research Overview

I am interested in testing the therapeutic potential of neural crest cells(NCC) derived from human embryonic stem cell(hESC), that can serve as a source for cell replacement therapy as well as a model for studying craniofacial disease progression. Research in my lab focuses on understanding molecular mechanisms regulating neural crest development using an innovative combination of multidisciplinary methodology (combining hESC differentiation with cutting-edge epigenomic and chromatin biology approaches and invivo embryological functional assays). Our goal of in vivo characterization of human enhancers that drive expression in subsets of cranial neural
crest cells allows us to isolate distinct craniofacial precursor cells, reveals gene regulatory mechanisms and their evolutionary conservation and serves as a powerful tool for genetic research on craniofacial development synergizing with the goals of fellow colleagues as well as the research community at large.
In addition, we are utilizing this developmental information to gain insight into the growth and metastasis of neural crest related tumors like neuroblastomas. Using an enhancer tagging approach, our attempts are focused on identifying and characterizing the tumor initiating cells and metastatic cells that reside within these extremely heterogeneous tumors. A comparative analysis of active enhancer profiles of various patient derived tumor lines and neural crest cells is underway.
We have pioneered and established methods for large scale, feeder free & serum free culture of hESC and their differentiation into early neural precursors as well as neural crest cells allowing for the characterization of these transient developmental cell populations at an unprecedented scale. We have also optimized lentiviral mediated
methodologies for genetically labeling, stable over-expression or inducible knockdown of genes of interest in hESC’s, cancer cells and developing embryos.

Experimental strategy used by our laboratory personnel include….
1) propagation of human pluripotent stem cells, their differentiation into NCC and culture of primary human NCC as well as neural crest related cancer cell lines in the lab. These cells are used for chromatin and RNA extraction followed by ChIP-seq and RNA seq analysis to identify the subset of epigenetically marked and developmentally regulated genes and their enhancers.
2) generation of lentivirally transduced hESC that express fluorescent reporters, shRNA for knockdown or cDNA’s of candidate epigenetic regulators of neural crest cells. The modified cells will be used for invitro assays, RNA-seq analysis and in ovo assays (transplantation of hESC/ NCC into the chick neural tube to follow development and differentiation of human neural crest cells in response to in ovo signaling cues).
3) generation of lentivirally transduced neuroblastoma cell lines to express fluorescent reporters, shRNA for knockdown or cDNA’s of candidate epigenetic regulators of neural crest cells in parallel and complementary studies to gain developmental insight into cancer progression. The modified cells will be used for RNA-seq, ChIP seq analysis; in vitro metastasis assays and in ovo (angiogenic, migration & metastasis) assays.
4) preparation of protein extracts from human cells or bacterial cultures for purification of macromolecular complexes for identification by mass spectrometry; protein functional assays; immunoprecipitation and western blot analysis.
5) testing hypothesis developed from cell culture assays in vivo using appropriate model organism( Xenopus embryos for morpholino mediated knockdown and overexpression to study the effect of candidate genes on NCC development; zebrafish/ Xenopus/ chick embryos for tol2 transposon based transgenesis assays for enhancer function; generation of tissue specific reporter mice using well characterized and validated enhancers).