Research & Scholarship
Current Research and Scholarly Interests
The long-term goal of our research is to understand the fundamental mechanisms that govern and reprogram cellular fate during development, regeneration and disease. We are specifically interested in-
1.Reprogramming approaches for musculoskeletal regeneration
Discovery of induced pluripotency by Yamanaka and colleagues has revolutionized the field of regenerative medicine. Induced pluripotent stem cells (iPSC), generated by introduction of a few defined factors in a somatic cell, provide an ideal patient-specific source for disease modeling, drug discovery and cellular therapies. Clinically, these findings have uncovered the possibility of unprecedented sources for patient-autologous cells with far reaching implications in a variety of diseases. From the basic biology perspective, these findings have revealed that cell fates are inherently plastic and are dynamically regulated. Our research is geared towards applying reprogramming approaches towards musculoskeletal regeneration especially cartilage regeneration that remains an unmet medical need.
2.Mechanisms underlying stem cell self-renewal, differentiation and cancer
We are interested in understanding the role of the extracellular matrix in regulating stem cell self-renewal and differentiation, and how this regulation goes awry in cancer. Understanding the acquisition and maintenance of the ‘differentiated’ state can provide important clues regarding the ‘dedifferentiation’ associated with cancer.
3.Epigenetic regulation in development and disease
DNA methylation is an epigenetic mark associated with long-term gene silencing during early development and lineage specification. The other side of the coin i.e. DNA demethylation has received scant attention over the years mainly due to the inability to identify enzymes that could mediate the removal of the methylation marks. Recent studies by our group and others have uncovered novel DNA repair based DNA demethylation pathways. Another exciting discovery is that of the ‘sixth base’ in DNA i.e. hydroxylation of methylated cytosines (5mC) by enzymes leading to ‘5hmC’ that is present in many tissues. The role and effect of 5hmC on 5mC turnover and hence DNA demethylation, on gene expression per se and stem cell fate and differentiation is a topic of vigorous interest. We are exploring the role of these novel DNA demethylation regulators in cartilage development, regeneration and disease. Our recent studies have uncovered a dysregulation of the DNA demethylation pathways in the widely prevalent age-associated disorder, Osteoarthritis. We are currently investigating the mechanistic details of these epigenetic pathways in Osteoarthritis.
Independent Studies (5)
- Directed Reading in Orthopedic Surgery
ORTHO 299 (Aut, Win, Spr, Sum)
- Early Clinical Experience in Orthopedic Surgery
ORTHO 280 (Aut, Win, Spr, Sum)
- Graduate Research
ORTHO 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
ORTHO 370 (Aut, Win, Spr, Sum)
- Undergraduate Research
ORTHO 199 (Aut, Win, Spr, Sum)
- Directed Reading in Orthopedic Surgery
Graduate and Fellowship Programs
A critical role for AID in the initiation of reprogramming to induced pluripotent stem cells
2013; 27 (3): 1107-1113
Mechanistic insights into the reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs) are limited, particularly for early acting molecular regulators. Here we use an acute loss of function approach to demonstrate that activation-induced deaminase (AID) activity is necessary for the initiation of reprogramming to iPSCs. While AID is well known for antibody diversification, it has also recently been shown to have a role in active DNA demethylation in reprogramming toward pluripotency and development. These findings suggested a potential role for AID in iPSC generation, yet, iPSC yield from AID-knockout mouse fibroblasts was similar to that of wild-type (WT) fibroblasts. We reasoned that an acute loss of AID function might reveal effects masked by compensatory mechanisms during development, as reported for other proteins. Accordingly, we induced an acute reduction (>50%) in AID levels using 4 different shRNAs and determined that reprogramming to iPSCs was significantly impaired by 79 ± 7%. The deaminase activity of AID was critical, as coexpression of WT but not a catalytic mutant AID rescued reprogramming. Notably, AID was required only during a 72-h time window at the onset of iPSC reprogramming. Our findings show a critical role for AID activity in the initiation of reprogramming to iPSCs.
View details for DOI 10.1096/fj.12-222125
View details for Web of Science ID 000315585200024
View details for PubMedID 23212122
Cathepsins L and Z Are Critical in Degrading Polyglutamine-containing Proteins within Lysosomes
JOURNAL OF BIOLOGICAL CHEMISTRY
2012; 287 (21): 17471-17482
In neurodegenerative diseases caused by extended polyglutamine (polyQ) sequences in proteins, aggregation-prone polyQ proteins accumulate in intraneuronal inclusions. PolyQ proteins can be degraded by lysosomes or proteasomes. Proteasomes are unable to hydrolyze polyQ repeat sequences, and during breakdown of polyQ proteins, they release polyQ repeat fragments for degradation by other cellular enzymes. This study was undertaken to identify the responsible proteases. Lysosomal extracts (unlike cytosolic enzymes) were found to rapidly hydrolyze polyQ sequences in peptides, proteins, or insoluble aggregates. Using specific inhibitors against lysosomal proteases, enzyme-deficient extracts, and pure cathepsins, we identified cathepsins L and Z as the lysosomal cysteine proteases that digest polyQ proteins and peptides. RNAi for cathepsins L and Z in different cell lines and adult mouse muscles confirmed that they are critical in degrading polyQ proteins (expanded huntingtin exon 1) but not other types of aggregation-prone proteins (e.g. mutant SOD1). Therefore, the activities of these two lysosomal cysteine proteases are important in host defense against toxic accumulation of polyQ proteins.
View details for DOI 10.1074/jbc.M112.352781
View details for Web of Science ID 000306373000047
View details for PubMedID 22451661
DNA Demethylation Dynamics
2011; 146 (6): 866-872
The discovery of cytosine hydroxymethylation (5hmC) suggested a simple means of demethylating DNA and activating genes. Further experiments, however, unearthed an unexpectedly complex process, entailing both passive and active mechanisms of DNA demethylation by the ten-eleven translocation (TET) and AID/APOBEC families of enzymes. The consensus emerging from these studies is that removal of cytosine methylation in mammalian cells can occur by DNA repair. These reports highlight that in certain contexts, DNA methylation is not fixed but dynamic, requiring continuous regulation.
View details for DOI 10.1016/j.cell.2011.08.042
View details for Web of Science ID 000295258100010
View details for PubMedID 21925312
Reprogramming towards pluripotency requires AID-dependent DNA demethylation
2010; 463 (7284): 1042-U57
Reprogramming of somatic cell nuclei to yield induced pluripotent stem (iPS) cells makes possible derivation of patient-specific stem cells for regenerative medicine. However, iPS cell generation is asynchronous and slow (2-3 weeks), the frequency is low (<0.1%), and DNA demethylation constitutes a bottleneck. To determine regulatory mechanisms involved in reprogramming, we generated interspecies heterokaryons (fused mouse embryonic stem (ES) cells and human fibroblasts) that induce reprogramming synchronously, frequently and fast. Here we show that reprogramming towards pluripotency in single heterokaryons is initiated without cell division or DNA replication, rapidly (1 day) and efficiently (70%). Short interfering RNA (siRNA)-mediated knockdown showed that activation-induced cytidine deaminase (AID, also known as AICDA) is required for promoter demethylation and induction of OCT4 (also known as POU5F1) and NANOG gene expression. AID protein bound silent methylated OCT4 and NANOG promoters in fibroblasts, but not active demethylated promoters in ES cells. These data provide new evidence that mammalian AID is required for active DNA demethylation and initiation of nuclear reprogramming towards pluripotency in human somatic cells.
View details for DOI 10.1038/nature08752
View details for Web of Science ID 000275108400028
View details for PubMedID 20027182
Nuclear reprogramming in heterokaryons is rapid, extensive, and bidirectional
2009; 23 (5): 1431-1440
An understanding of nuclear reprogramming is fundamental to the use of cells in regenerative medicine. Due to technological obstacles, the time course and extent of reprogramming of cells following fusion has not been assessed to date. Here, we show that hundreds of genes are activated or repressed within hours of fusion of human keratinocytes and mouse muscle cells in heterokaryons, and extensive changes are observed within 4 days. This study was made possible by the development of a broadly applicable approach, species-specific transcriptome amplification (SSTA), which enables global resolution of transcripts derived from the nuclei of two species, even when the proportions of species-specific transcripts are highly skewed. Remarkably, either phenotype can be dominant; an excess of primary keratinocytes leads to activation of the keratinocyte program in muscle cells and the converse is true when muscle cells are in excess. We conclude that nuclear reprogramming in heterokaryons is rapid, extensive, bidirectional, and dictated by the balance of regulators contributed by the cell types.
View details for DOI 10.1096/fj.08-122903
View details for Web of Science ID 000266651700019
View details for PubMedID 19141533