Research Centers

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Rajagopal Lab

Our laboratory focuses on organ regeneration and the application of developmental biology to human disease. We focus on the lung as a model system since there is an abundance of respiratory diseases of unknown cause without cures.

Jayaraj Rajagopal, MD
Physician, Pulmonary and Critical Care Unit, Massachusetts General Hospital
Instructor in Medicine, Harvard Medical School

Protocols:
Information Related to Cell Lines

Protocol for generating tracheospheres from iPS cells using engraftment into an immunodeficient mouse

Protocol for human ES and iPS Cell differentiation to NKx2 1+cells

Protocol for mouse ES cell differentiation to Nkx2 1+cells

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The lung is a vertebrate invention that allowed the early tetrapods to leave the water and colonize the land. Oxygen is the essential actor in aerobic metabolism and the lung’s cardinal function is to mediate the efficient transfer of oxygen from theatmosphere to the circulatory system. Given the lung’s essential function, diseases of the lung are often life-threatening or incapacitating.

 

We seek to isolate and culture lung stem cells and to understand their role in normal lung epithelial homeostasis and in regeneration after tissue injury. Lung stem cells purified from a donor animal or obtained from embryonic stem cell culture will provide novel reagents for generating in vitro models of human lung disease. We are also exploring the in vivo engraftment of lung epithelial stem cells as a basis for regenerative medical therapies. Our inquiries into the mechanisms underlying embryonic lung development and adult lung epithelial regeneration serve as a framework within which we seek to understand how the reactivation and distortion of normal developmental processes results in human lung disease.

In a parallel effort, we study the murine trachea as a robust model of organ regeneration. We seek to answer longstanding historical questions in regeneration biology using this highly reproducible, rapid experimental model. Our goals include:

 

  1. Identifying the signal that initiates regeneration, as well as the signal that causes regeneration to stop (perhaps abnormally regulated in dysplastic precancerous tissues).
  2. Defining the mechanism that controls a regenerate’s size.
  3. Clarifying whether dedifferentiation and "spontaneous" developmental reprogramming is involved in normal epithelial restoration.
  4. Exploring whether cellular reprogramming can be used to produce lung progenitor cells from differentiated epithelial cells, which might be useful for in situ lung regeneration in diseases characterized by gross loss of cell components, for example, the wholesale loss of alveoli in emphysema.
  5. Isolating the lung stem cells that are responsible for airway regeneration so that they can form the basis of cellular therapies for diseases in which endogenous cells are genetically abnormal, as in Cystic Fibrosis.


This work will culminate in the identification of cells capable of carrying out regeneration, but it will also teach us how to modulate these cells for human regenerative therapies. As an example, we have already identified the Notch pathway as a regulator of mucous metaplasia in the embryonic and adult tracheal epithelium. Indeed, Notch antagonists can block human mediators of allergic asthma and prevent mucous metaplasia of the airways.

View Previous Research

Postdoctoral Fellows

Hongmei Mou, PhD
Ana Pardo, PhD
Tata Rao, PhD
Manju Shivaraju, PhD
Vladimir Vinarsky, MD
Rui Zhao, PhD 

Collaborators

Andrew Brack, PhD
Fernando Camargo, PhD
Chad Cowan, PhD
Konrad Hochedlinger, PhD
Douglas Melton, PhD
Kiran Musunuru, MD PhD
Lee Rubin, PhD
Richard Sherwood, PhD
Gary Tearney, MD PhD
Qiao Zhou, PhD

Graduate StudentsJorge VilloriaUndergraduates

Ryan Chow
Brandon Law
Jonathon Tran

Alumni

Andrea Brettler, Tim Fallon, Adam Lam, Mythili Prabhu





Current Research Interests

Identification of a lung stem cell

There are no definitively characterized lung stem cells. We are now prospectively identifying andculturing candidate lung stem cells. In the case of the hematopoietic system, the development of bone marrow irradiation was the key to the identification of hematopoietic stem cells. By analogy, we have developed an in vivo functional engraftment assay to characterize the full spectrum of differentiation of FACS-isolated tracheal progenitor cells.A variety of techniques including label-retention assays and in vivo single cell lineage analysis will be used to identify and sort putative stem cells from both normal and regenerating tracheal epithelium. We have already identified a number of novel cell surface markers that would be suitable to sort subpopulations of mouse tracheal epithelium.

Developing an In Vivo Functional Assay for Lung Stem Cells

The adult trachea is known to display continual cell turnover during normal homeostasis. In addition, the tracheal and airway epithelium are known to heal after toxic and thermal exposures. In aggregate, this phenomenology suggested that the trachea is capable of clinically relevant regeneration. Using an orthotopic tracheal transplantation model (Figure 1A) in which an injured trachea is grafted onto the trachea of a donor animal, we have developed an in vivo model of regeneration. Using genetically labeled donor and recipient cells, we demonstrated that regeneration is a local phenomenon mediated by rare residual cells that survive at the sites of injury (Figure 1B). These cells possess markers that suggest they are tracheal basal cells, and they initiate regeneration through a process of rapid proliferation. This is then followed by a well-orchestrated cascade of cellular differentiation that recapitulates the orderly progression of marker maturation seen in the embryo during the initial generation of tracheal epithelium. We next sought to identify the precise cellular origins of the progenitor cells which mediate this regenerative phenomenon. To do so, we developed a functional model of tracheal cell engraftment. Initially, using frozen and thawed trachea, we showed that cells resident in a host animal’s trachea can repopulate a tracheal “husk.” While cells that mediate normal regeneration act locally at the site of injury, they can be induced to spread and migrate over a suitably prepared recipient substrate (Figure 1B). By using a frozen and thawed esophagus, we have shown that tracheal cell identity and regeneration is independent of basement membrane and mesenchyme. This result focused all of our attention on the information present within airway epithelial cells themselves. As an extension of this work, we have engrafted dispersed singe-cell preparations of tracheal epithelium onto donor trachea in an adoptive transfer experiment and we subsequently demonstrate full epithelial reconstitution (Figure 1C). Quantitative repopulation experiments are underway. The question of the precise cell of origin of the regenerative phenomenon remains uncertain, but certainly suggests the possibility of a bona fide stem cell in the proximal airway.




Using developmental biology to dissect lung disease

Our work on lung developmental biology and organ regeneration is the biological platform from which we attempt to cure diseases of the respiratory tract in man. Many diseases of the lung are characterized by abnormal cellular proliferation and differentiation. Diseases of the airways such as asthma, bronchitis, and Cystic Fibrosis are all characterized by mucous metaplasia, in which there is both an aberrant epithelial hyperplasia and an excess of mucous cells. On the other hand, Idiopathic Pulmonary Fibrosis (IPF) and radiation-induced fibrosis are characterized by the aberrant proliferation of mesenchyme cells of unclear identity and origin. Similarly, cancer is fundamentally an unrestrained proliferation of both tumor epithelium and mesenchyme.Dissecting mouse models of human lung disease

We employ modern mouse genetics and in vivo lineage tracing to study lung organogenesis and mouse models of human disease. This will enable us to identify the 'cells of origin' in diseases such as pulmonary fibrosis and lung cancer. Future studies will aim to identify and manipulate the specific signaling cascades that drive the pathologic cell behaviors that produce abnormal tissue responses in disease. This work will, in turn, form the basis for chemical screens designed to discover new drug targets that act by inhibiting these pathways.

Human developmental biology applied to lung disease

In parallel with our laboratory-based approaches, we have also established collaborations with clinicians, clinician-scientists, and human tissue banks in order to directly study human disease-causing cells. Using laser capture micro dissection of well defined populations of lung cells, we will identify unique markers for various disease-associated cell types. This will begin with a comprehensive systems level analysis of the transcriptome of each specific population of disease-associated lung cells. In collaboration with the Broad Institute, these pathways will then be compared to the genome-wide data we have already assembled on the transcriptional networks that orchestrate mouse embryonic lung development. This will be complemented by a computational analysis of data derived from mining publicly available lung disease microarray datasets. The computationally identified networks are then used to elucidate the specific transcriptional and signaling pathways that result in aberrant proliferation and differentiation of human cells in both human tissue culture and mouse xenograft models. Lineage analysis will be performed on these human models themselves, to directly complement our lineage studies in mouse models of human disease.


Human model systems of lung disease: airway stem cells from induced-pluripotent stem cells

We are currently optimizing the production of airway progenitor cells from human induced-pluripotent stem (iPS) cells that are derived from the skin fibroblasts of patients with various lung diseases.  The production of embryonic endoderm from hES cells has already been optimized.  To optimize the differentiation and amplification of airway progenitor cells from endoderm derived from pluripotent stem cells, we are using high throughput chemical and biological screening platforms on genetically modified iPS cells which signal lung identity using fluorescent reporters of lung differentiation (SP-C-gfp, CC10-gfp, and Nkx2.1-gfp). 

Generating In Vitro Cellular and Explant Models of Human Airway Disease

We have succeeded in growing airway epithelial cultures and regenerating tracheal explants in vitro.  Various systems that permit the clonal analysis of progenitor cells and their progeny are now being deployed in vitro on tracheal explants.  Screenings for chemical compounds that promote or inhibit cell proliferation and differentiation in our explant system is also underway.  Such screening may identify novel signaling factors that result in mucous metaplasia or hyperplasia.  Screening on human tissues will complement the work done in mouse.  Screening of diseased human airway epithelium (such as asthmatic airway) should offer the possibility of identifying specific drug targets for specific diseases.  Intriguingly, the application of Notch to regenerating tracheal explants produces an in vitro model of mucous metaplasia, the cardinal pathology associated with proximal airway diseases including bronchitis, bronchiectasis, asthma, and Cystic Fibrosis.  Further chemical screening of such metaplastic tracheal explants should allow for the screening of therapeutically relevant drugs to be used for inhalational therapy in these disorders.  In fact, we have also begun to explore the utility of inhaled gamma-secretase inhibitors in an animal model of allergic airways disease.

Cell therapies for lung disease: Curing Cystic Fibrosis with airway progenitors

 

We have chosen to focus on Cystic Fibrosis (CF) as a prototypic lung disease that can be studied, and eventually treated using stem cell-derived therapies.  We have done so because CF is the most common monogenetic disorder of the lung, and because it affects the young, often resulting in premature death.  CF is characterized by repeated bouts of pneumonia which, over time, cause the airway and lung architecture to become grossly distorted, precipitating further infection.  This vicious cycle of infection and injury ultimately results in death.  The cause of this phenotypically complicated disease is genetically rather simple: loss-of-function mutations in the Cystic Fibrosis Transmembrane Regulator (CFTR), an epithelial chloride channel.  The loss of chloride secretion eventually leads to the production of abnormal airway surface fluid which then results in abnormally viscous mucous and infection.  By targeting the mutant CFTR locus in iPS cells produced from CF patients, we will replace the defective disease-causing CFTR mutation with a normal gene.  As described above, these corrected CF-iPS cells and their disease-bearing counterparts will be used to produce airway progenitor cells.  These iPS-derived airway progenitor cells can then serve as human model systems to test novel CF therapies and to further dissect the cellular and tissue level mechanisms that contribute to the CF phenotype.  The use of patient-specific human cells circumvents the lack of phenotype in mouse models of CFTR deletion.  Furthermore, genetically rescued CF iPS cells can themselves be used as patient-matched cell replacement therapies.  We have already developed strategies for in situ engraftment of airway stem cells and are refining and optimizing these techniques in preparation for future clinical applications. 

 

In parallel with our laboratory-based approaches, we have also established collaborations with clinicians, clinician-scientists, and human tissue banks in order to directly study human disease-causing cells. Using laser capture micro dissection of well defined populations of lung cells, we will identify unique markers for various disease-associated cell types. This will begin with a comprehensive systems level analysis of the transcriptome of each specific population of disease-associated lung cells. In collaboration with the Broad Institute, these pathways will then be compared to the genome-wide data we have already assembled on the transcriptional networks that orchestrate mouse embryonic lung development. This will be complemented by a computational analysis of data derived from mining publicly available lung disease microarray datasets. The computationally identified networks are then used to elucidate the specific transcriptional and signaling pathways that result in aberrant proliferation and differentiation of human cells in both human tissue culture and mouse xenograft models. Lineage analysis will be performed on these human models themselves, to directly complement our lineage studies in mouse models of human disease.

The Rajagopal lab is currently hiring for a Postdoctoral Fellow:  http://www.hsci.harvard.edu/jobs/postdoctoral-fellow-3

To apply for a position within the Rajagopal Laboratory, please email your current CV and 3 letters of references to Dr. Jayaraj Rajagopal.

Mass General Researchers Take Major Step Toward a Treatment for Cystic Fibrosis

Researchers in the Center for Regenerative Medicine at Massachusetts General Hospital and the Harvard Stem Cell Institute have taken a critical step toward a treatment for Cystic fibrosis and other fatal lung diseases.

Rajagopal J, Doudna J, and Szostak J. Stereochemical Course of Catalysis by the Tetrahymena Ribozyme. Science. 1989;244:692-94.

Coutre S, Ellington A, Gerber A, Cherry J, Doudna J, Green R, Hanna M, Rajagopal J, Szostak J. Mutational Analysis of Conserved Nucleotides in a Self-splicing Group I Intron. Journal of Molecular Biology. 1990;215:345-58.

Rajagopal J, Anderson W, Kume S, Martinez O, Melton D. Insulin Staining of ES Cell Progeny from Insulin Uptake. Science. 2003;299:363.

Hanson M, Madsen O, Semb H, Rajagopal J, Melton D, Serup P. Artifactual Insulin Release from Differentiated Embryonic Stem Cells. Diabetes. 2004;53:2603-9.

Zhou Q, Law AC, Rajagopal J, Anderson WJ, Gray PA, Melton DA. A Multipotent Progenitor Domain Guides Pancreatic Organogenesis. Dev Cell. 2007;13:103-14.

Tu X, Joeng KS, Nakayama KI, Rajagopal J, Carroll TJ, McMahon AP, Long F. Noncanonical Wnt signaling through G protein-linked PKCdelta activation promotes bone formation. Dev Cell. 2007;12:113-27.

Shaw AT, Meissner A, Dowdle JA, Crowley D, Magendatz M, Ouyang C, Parisi T, Rajagopal J, Blank LJ, Bronson RT, Stone JR, Tuveson DA, Jaenisch R, Jacks T. Sprouty- regulates oncogenic K-ras in lung development and tumorigenesis. Genes Dev. 2008;15:694-707.

Anderson WJ, Zhou Q, Alcalde V, Kaneko OF, Blank LJ, Sherwood RI, Guseh JS, Rajagopal J, Melton DA. Genetic targeting of the endoderm with claudin-6CreER. Dev Dyn. 2008;237:504-12.

Rajagopal J, Carroll TJ, Guseh JS, Bores SA, Blank LJ, Anderson WJ, Yu J, Zhou Q, McMahon AP, Melton DA. Wnt7b stimulates embryonic lung growth by coordinately increasing the replication of epithelium and mesenchyme. Development. 2008;135: 1625-34.

Zhou Q, Brown J, Kanarek A, Rajagopal J, Melton DA. In vivo reprogramming of adult pancreatic exocrine cells to beta-cells. Nature. 2008; 455: 627-32.

Yu,J, Carroll TJ, Rajagopal J, Kobayashi A, Ren Q, McMahon AP. A Wnt7b-dependent pathway regulates the orientation of epithelial cell division and establishes the cortico-medullary axis of the mammalian kidney. Development. 2008; 136: 161-171.

Stenman JM, Rajagopal J, Carroll TJ, Ishibashi M, McMahon J, McMahon AP. Canonical Wnt signaling regulates organ-specific assembly and differentiation of CNS vasculature. Science. 2008; 322: 1247-50.

Guseh J, Bores S, Stanger B, Zhou Q, Anderson W, Melton D, Rajagopal J. Notch signaling regulates epithelial cell fate in both embryonic lung and adult airway epithelium. Development, Development. 2009 May;136(10):1751-9. Epub 2009 Apr 15.

Mou H, Zhao R, Sherwood R, Ahfeldt T, Lapey A, Wain J, Sicilian L, Izvolsky K, Musunuru K, Cowan C, Rajagopal J.  Generation of Multipotent Lung and Airway Progenitors from Mouse ESCs and Patient-Specific Cystic Fibrosis iPSCs.  Cell Stem Cell. 2012 Apr 6;10(4):385-97.

Longmire TA, Ikonomou L, Hawkins F, Christodoulou C, Cao Y, Jean JC, Kwok LW, Mou H, Rajagopal J, Shen SS, Dowton AA, Serra M, Weiss DJ, Green MD, Snoeck HW, Ramirez MI, Kotton DN.  Efficient derivation of purified lung and thyroid progenitors from embryonic stem cells.  Cell Stem Cell. 2012 Apr 6;10(4):398-411.

Mou H, Zhao R, Sherwood R, Ahfeldt T, Lapey A, Wain J, Sicilian L, Izvolsky K, Musunuru K, Cowan C, Rajagopal J. Generation of multipotent lung and airway progenitors from mouse ESCs and patient-specific cystic fibrosis iPSCs. Cell Stem Cell. 2012 Apr 6;10(4):385-97.

Kim JK, Vinarsky V, Wain JC, Zhao R, Jung K, Choi J, Lam A, Pardo-Saganta A, Breton S, Rajagopal J, Yun SH. In vivo imaging of tracheal epithelial cells in mice during airway regeneration.  Am J Respir Cell Mol Biol. 2012; 47: 864-868.

Muzykewicz DA, Black ME, Muse V, Numis AL, Rajagopal J, Thiele EA, Sharma A. Multifocal micronodular pneumocyte hyperplasia: computed tomographic appearance and follow-up in tuberous sclerosis complex. J Comput Assist Tomogr. 2012 Sep-Oct;36(5):518-22. PubMed PMID: 22992599.

Pardo-Saganta A, Law BM, Gonzalez-Celeiro M, Vinarsky V, Rajagopal J. Ciliated cells of the pseudostratified airway epithelium do not become mucous cells after OVA challenge. Am J Respir Cell Mol Biol. 2013; Mar: 48(3): 364-373.  PMCID: PMC3604083.

Watanabe H, Francis JM, Woo MS, Lin W, Fries DF, Peng S, Snyder EL, Tata PR, Izzo F, Schinzel AC, Cho J, Hammerman PS, Verhaak RG, Hahn WC, Rajagopal J, Jacks T, Meyerson M. Integrated cistromic and expression analysis of amplified NKX2.1 in lung adenocarcinoma identifies LMO3 as a functional transcriptional target.  Genes and Development. 2013; Jan 15:27(2): 197-210.

Tata PR, Pardo-Saganta A, Prabhu M, Vinarsky V, Law BM, Fontaine BA, Tager AM, Rajagopal J. Airway Specific Inducible Transgene Expression Using Aerosolized Doxycycline. Am J Respir Cell Mol Biol. 2013; Dec: 49(6): 1048-1056. PubMed PMID: 23848320.

Weiss DJ, Bates JH, Gilbert T, Liles WC, Lutzko C, Rajagopal J, Prockop DJ. Conference Report: Stem Cells and Cell Therapies in Lung Biology and Diseases University of Vermont, July 2011. Ann Am Thorac Soc. 2013; 10: S25-44. PubMed PMID: 23869447.

Tata PR, Mou H, Pardo-Saganta A, Zhao R, Prabhu M, Law BM, Vinarsky V, Cho JL, Breton S, Sahay A, Medoff BD, Rajagopal J. Dedifferentiation of committed luminal epithelial cells into functional stem cells in vivo. Nature, 2013; 503; 218-223 (highlighted in Nature News and Views and featured in Cell Leading Edge Select where a paper from another journal is reviewed as well as in a Cell Stem Cell preview, and selected as an F1000 Prime article).

Guha A, Vasconcelos M, Zhao R, Gower AC, Rajagopal J, Cardoso WV. Analysis of Notch signaling-dependent gene expression in developing airways reveals diversity of Clara cells. PLoS One. 2014 Feb 21;9(2):e88848. doi: 10.1371/journal.pone.0088848. eCollection 2014. PMID: 24586412.

Zhao R, Fallon TR, Saladi SV, Pardo-Saganta A, Villoria J, Mou H, Vinarsky V, Gonzalez-Celeiro M, Nunna N, Hariri LP, Camargo F, Ellisen LW, Rajagopal J. Yap Tunes Airway Epithelial Size and Architecture by regulating the Identity, maintenance, and self-renewal of stem cells. Developmental Cell, in press.