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.
- Orthopaedic Tissue Engineering
ORTHO 270 (Win)
Independent Studies (9)
- Directed Reading in Cancer Biology
CBIO 299 (Win, Spr)
- Directed Reading in Orthopedic Surgery
ORTHO 299 (Aut, Sum)
- Early Clinical Experience in Orthopedic Surgery
ORTHO 280 (Aut, Sum)
- Graduate Research
CBIO 399 (Aut, Win, Spr, Sum)
- Graduate Research
ORTHO 399 (Aut, Sum)
- Medical Scholars Research
ORTHO 370 (Aut, Sum)
- Out-of-Department Advanced Research Laboratory in Experimental Biology
BIO 199X (Aut, Win, Spr)
- Teaching in Cancer Biology
CBIO 260 (Spr)
- Undergraduate Research
ORTHO 199 (Aut, Win, Spr, Sum)
- Directed Reading in Cancer Biology
Prior Year Courses
- Orthopaedic Tissue Engineering
ORTHO 270 (Win)
- Orthopaedic Tissue Engineering
Postdoctoral Faculty Sponsor
Graduate and Fellowship Programs
Identification of Human Juvenile Chondrocyte-Specific Factors that Stimulate Stem Cell Growth
TISSUE ENGINEERING PART A
2016; 22 (7-8): 645-653
Although regeneration of human cartilage is inherently inefficient, age is an important risk factor for osteoarthritis. Recent reports have provided compelling evidence that juvenile chondrocytes (from donors below 13 years of age) are more efficient at generating articular cartilage as compared to adult chondrocytes. However, the molecular basis for such a superior regenerative capability is not understood. To identify the cell-intrinsic differences between juvenile and adult cartilage, we have systematically profiled global gene expression changes between a small cohort of human neonatal/juvenile and adult chondrocytes. No such study is available for human chondrocytes although young and old bovine and equine cartilage have been recently profiled. Our studies have identified and validated new factors enriched in juvenile chondrocytes as compared to adult chondrocytes including secreted extracellular matrix factors chordin-like 1 (CHRDL1) and microfibrillar-associated protein 4 (MFAP4). Network analyses identified cartilage development pathways, epithelial-mesenchymal transition, and innate immunity pathways to be overrepresented in juvenile-enriched genes. Finally, CHRDL1 was observed to aid the proliferation and survival of bone marrow-derived human mesenchymal stem cells (hMSC) while maintaining their stem cell potential. These studies, therefore, provide a mechanism for how young cartilage factors can potentially enhance stem cell function in cartilage repair.
View details for DOI 10.1089/ten.tea.2015.0366
View details for Web of Science ID 000374761600007
View details for PubMedID 26955889
Stable 5-Hydroxymethylcytosine (5hmC) Acquisition Marks Gene Activation During Chondrogenic Differentiation
JOURNAL OF BONE AND MINERAL RESEARCH
2016; 31 (3): 524-534
Regulation of gene expression changes during chondrogenic differentiation by DNA methylation and demethylation is little understood. Methylated cytosines (5mC) are oxidized by the ten-eleven-translocation (TET) proteins to 5-hydroxymethylcytosines (5hmC), 5-formylcytosines (5fC) and 5-carboxylcytosines (5caC) eventually leading to a replacement by unmethylated cytosines (C) i.e. DNA demethylation. Additionally, 5hmC is stable and acts as an epigenetic mark by itself. Here, we report that global changes in 5hmC mark chondrogenic differentiation in vivo and in vitro. Tibia anlagen and growth plate analyses during limb development at mouse embryonic days E 11.5, 13.5 and 17.5 showed dynamic changes in 5hmC levels in the differentiating chondrocytes. A similar increase in 5hmC levels was observed in the ATDC5 chondroprogenitor cell line accompanied by increased expression of the TET proteins during in vitro differentiation. Loss of TET1 in ATDC5 decreased 5hmC levels and impaired differentiation, demonstrating a functional role for TET1-mediated 5hmC dynamics in chondrogenic differentiation. Global analyses of the 5hmC-enriched sequences during early and late chondrogenic differentiation identified 5hmC distribution to be enriched in the regulatory regions of genes preceding the transcription start site (TSS) as well as in the gene bodies. Stable gains in 5hmC were observed in specific subsets of genes including genes associated with cartilage development and in chondrogenic lineage-specific genes. 5hmC gains in regulatory promoter and enhancer regions as well as in gene bodies were strongly associated with activated but not repressed genes, indicating a potential regulatory role for DNA hydroxymethylation in chondrogenic gene expression. This article is protected by copyright. All rights reserved.
View details for DOI 10.1002/jbmr.2711
View details for Web of Science ID 000373596800006
View details for PubMedID 26363184
The first international workshop on the epigenetics of osteoarthritis.
Connective tissue research
Osteoarthritis (OA) is a major clinical problem across the world, in part due to the lack of disease-modifying drugs resulting, to a significant degree, from our incomplete understanding of the underlying molecular mechanisms of the disease. Emerging evidence points to a role of epigenetics in the pathogenesis of OA, but research in this area is still in its early stages. In order to summarize current knowledge and to facilitate the potential coordination of future research activities, the first international workshop on the epigenetics of OA was held in Amsterdam in October 2015. Recent findings on DNA methylation and hydroxymethylation, histone modifications, noncoding RNAs, and other epigenetic mechanisms were presented and discussed. The workshop demonstrated the advantage of bringing together those working in this nascent field and highlights from the event are summarized in this report in the form of summaries from invited speakers and organizers.
View details for DOI 10.3109/03008207.2016.1168409
View details for PubMedID 27028588
3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.
View details for DOI 10.3791/53085
View details for Web of Science ID 000368572800032
View details for PubMedID 26484414
Early induction of a prechondrogenic population allows efficient generation of stable chondrocytes from human induced pluripotent stem cells
2015; 29 (8): 3399-3410
Regeneration of human cartilage is inherently inefficient; an abundant autologous source, such as human induced pluripotent stem cells (hiPSCs), is therefore attractive for engineering cartilage. We report a growth factor-based protocol for differentiating hiPSCs into articular-like chondrocytes (hiChondrocytes) within 2 weeks, with an overall efficiency >90%. The hiChondrocytes are stable and comparable to adult articular chondrocytes in global gene expression, extracellular matrix production, and ability to generate cartilage tissue in vitro and in immune-deficient mice. Molecular characterization identified an early SRY (sex-determining region Y) box (Sox)9(low) cluster of differentiation (CD)44(low)CD140(low) prechondrogenic population during hiPSC differentiation. In addition, 2 distinct Sox9-regulated gene networks were identified in the Sox9(low) and Sox9(high) populations providing novel molecular insights into chondrogenic fate commitment and differentiation. Our findings present a favorable method for generating hiPSC-derived articular-like chondrocytes. The hiChondrocytes are an attractive cell source for cartilage engineering because of their abundance, autologous nature, and potential to generate articular-like cartilage rather than fibrocartilage. In addition, hiChondrocytes can be excellent tools for modeling human musculoskeletal diseases in a dish and for rapid drug screening.-Lee, J., Taylor, S. E. B., Smeriglio, P., Lai, J., Maloney, W. J., Yang, F., Bhutani, N. Early induction of a prechondrogenic population allows efficient generation of stable chondrocytes from human induced pluripotent stem cells.
View details for DOI 10.1096/fj.14-269720
View details for Web of Science ID 000358796900027
- Genome-Wide Mapping of DNA Hydroxymethylation in Osteoarthritic Chondrocytes ARTHRITIS & RHEUMATOLOGY 2015; 67 (8): 2129-2140
Collagen VI Enhances Cartilage Tissue Generation by Stimulating Chondrocyte Proliferation
TISSUE ENGINEERING PART A
2015; 21 (3-4): 840-849
Regeneration of human cartilage is inherently inefficient. Current cell-based approaches for cartilage repair, including autologous chondrocytes, are limited by the paucity of cells, associated donor site morbidity, and generation of functionally inferior fibrocartilage rather than articular cartilage. Upon investigating the role of collagen VI (Col VI), a major component of the chondrocyte pericellular matrix (PCM), we observe that soluble Col VI stimulates chondrocyte proliferation. Interestingly, both adult and osteoarthritis chondrocytes respond to soluble Col VI in a similar manner. The proliferative effect is, however, strictly due to the soluble Col VI as no proliferation is observed upon exposure of chondrocytes to immobilized Col VI. Upon short Col VI treatment in 2D monolayer culture, chondrocytes maintain high expression of characteristic chondrocyte markers like Col2a1, agc, and Sox9 whereas the expression of the fibrocartilage marker Collagen I (Col I) and of the hypertrophy marker Collagen X (Col X) is minimal. Additionally, Col VI-expanded chondrocytes show a similar potential to untreated chondrocytes in engineering cartilage in 3D biomimetic hydrogel constructs. Our study has, therefore, identified soluble Col VI as a biologic that can be useful for the expansion and utilization of scarce sources of chondrocytes, potentially for autologous chondrocyte implantation. Additionally, our results underscore the importance of further investigating the changes in chondrocyte PCM with age and disease and the subsequent effects on chondrocyte growth and function.
View details for DOI 10.1089/ten.tea.2014.0375
View details for Web of Science ID 000349611600039
Comparative Potential of Juvenile and Adult Human Articular Chondrocytes for Cartilage Tissue Formation in Three-Dimensional Biomimetic Hydrogels
TISSUE ENGINEERING PART A
2015; 21 (1-2): 147-155
Regeneration of human articular cartilage is inherently limited and extensive efforts have focused on engineering the cartilage tissue. Various cellular sources have been studied for cartilage tissue engineering including adult chondrocytes, as well as embryonic or adult stem cells. Juvenile chondrocytes (from donors below 13 years of age) have recently been reported to be a promising cell source for cartilage regeneration. Previous studies have compared the potential of adult and juvenile chondrocytes or adult and osteoarthritic (OA) chondrocytes. To comprehensively characterize the comparative potential of young, old and diseased chondrocytes, here we examined cartilage formation by juvenile, adult and OA chondrocytes in 3D biomimetic hydrogels composed of poly(ethylene glycol) and chondroitin sulfate. All three human articular chondrocytes were encapsulated in the 3D biomimetic hydrogels and cultured for 3 or 6 weeks to allow maturation and extracellular matrix formation. Outcomes were analyzed using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. After 3 and 6 weeks, juvenile chondrocytes showed a greater upregulation of chondrogenic gene expression than adult chondrocytes, while OA chondrocytes showed a downregulation. Aggrecan and type II collagen deposition and GAG accumulation were high for juvenile and adult chondrocytes but not for OA chondrocytes. Similar trend was observed in the compressive moduli of the cartilage constructs generated by the three different chondrocytes. In conclusion, the juvenile, adult and OA chondrocytes showed differential responses in the 3D biomimetic hydrogels. The 3D culture model described here may also provide a useful tool to further study the molecular differences among chondrocytes from different stages, which can help elucidate the mechanisms for age-related decline in the intrinsic capacity for cartilage repair.
View details for DOI 10.1089/ten.tea.2014.0070
View details for Web of Science ID 000348704900015
- A Global Increase in 5-Hydroxymethylcytosine Levels Marks Osteoarthritic Chondrocytes ARTHRITIS & RHEUMATOLOGY 2014; 66 (1): 90-100
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
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