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Forward Genetics in Cryptosporidium Enabled by Complete in Vitro Development in Stem Cell-Derived Intestinal Epithelium

76 Pages Posted: 8 Feb 2019 Sneak Peek Status: Published

See all articles by Georgia Wilke

Georgia Wilke

Washington University in St. Louis - Department of Molecular Microbiology

Lisa Funkhouser-Jones

Washington University in St. Louis - Department of Molecular Microbiology

Yi Wang

Washington University in St. Louis - Department of Pathology and Immunology

Soumya Ravindran

Washington University in St. Louis - Department of Molecular Microbiology

Qiuling Wang

Washington University in St. Louis - Department of Molecular Microbiology

Wandy L. Beatty

Washington University in St. Louis - Department of Molecular Microbiology

Megan T. Baldridge

Washington University in St. Louis - Division of Infectious Diseases

Kelli L. VanDussen

Washington University in St. Louis - Department of Pathology and Immunology

Bang Shen

Washington University in St. Louis - Department of Molecular Microbiology

Mark S. Kuhlenschmidt

University of Illinois at Urbana-Champaign - Department of Pathobiology

Theresa B. Kuhlenschmidt

University of Illinois at Urbana-Champaign - Department of Pathobiology

William H. Witola

University of Illinois at Urbana-Champaign - Department of Pathobiology

Thaddeus S. Stappenbeck

Washington University in St. Louis - Department of Pathology and Immunology

L. David Sibley

Washington University in St. Louis - Department of Molecular Microbiology

More...

Abstract

Despite being a frequent cause of severe diarrheal disease in infants and an opportunistic infection in immunocompromised patients, Cryptosporidium research has lagged due to a lack of facile culture methods. Here, we describe a platform for complete life cycle development and long-term growth of C. parvum in vitro using “air-liquid interface” (ALI) cultures derived from intestinal epithelial stem cells. Transcriptomic profiling revealed that differentiating epithelial cells grown under ALI conditions undergo profound changes in metabolism and development that enable complete development of the parasite life cycle in vitro. ALI cultures supported parasite expansion >100-fold and led to the production of viable oocysts that were transmissible in vitro and to mice. Transgenic parasite lines created using CRISPR/Cas9 were used to complete a genetic cross in vitro, demonstrating conventional meiosis and Mendelian segregation. ALI culture provides an accessible model that will enable innovative studies into Cryptosporidium biology and host interactions.

Keywords: stem cell, metabolism, differentiation, meiosis, forward genetics, enteric pathogen, parasitology

Suggested Citation

Wilke, Georgia and Funkhouser-Jones, Lisa and Wang, Yi and Ravindran, Soumya and Wang, Qiuling and Beatty, Wandy L. and Baldridge, Megan T. and VanDussen, Kelli L. and Shen, Bang and Kuhlenschmidt, Mark S. and Kuhlenschmidt, Theresa B. and Witola, William H. and Stappenbeck, Thaddeus S. and Sibley, L. David, Forward Genetics in Cryptosporidium Enabled by Complete in Vitro Development in Stem Cell-Derived Intestinal Epithelium (February 8, 2019). Available at SSRN: https://ssrn.com/abstract=3331307 or http://dx.doi.org/10.2139/ssrn.3331307
This is a paper under consideration at Cell Press and has not been peer-reviewed.

Georgia Wilke (Contact Author)

Washington University in St. Louis - Department of Molecular Microbiology

One Brookings Drive
Campus Box 1208
Saint Louis, MO 63130-4899
United States

Lisa Funkhouser-Jones

Washington University in St. Louis - Department of Molecular Microbiology ( email )

One Brookings Drive
Campus Box 1208
Saint Louis, MO 63130-4899
United States

Yi Wang

Washington University in St. Louis - Department of Pathology and Immunology

One Brookings Drive
Campus Box 1208
Saint Louis, MO 63130-4899
United States

Soumya Ravindran

Washington University in St. Louis - Department of Molecular Microbiology ( email )

One Brookings Drive
Campus Box 1208
Saint Louis, MO 63130-4899
United States

Qiuling Wang

Washington University in St. Louis - Department of Molecular Microbiology ( email )

One Brookings Drive
Campus Box 1208
Saint Louis, MO 63130-4899
United States

Wandy L. Beatty

Washington University in St. Louis - Department of Molecular Microbiology ( email )

One Brookings Drive
Campus Box 1208
Saint Louis, MO 63130-4899
United States

Megan T. Baldridge

Washington University in St. Louis - Division of Infectious Diseases

One Brookings Drive
Campus Box 1208
Saint Louis, MO 63130-4899
United States

Kelli L. VanDussen

Washington University in St. Louis - Department of Pathology and Immunology

One Brookings Drive
Campus Box 1208
Saint Louis, MO 63130-4899
United States

Bang Shen

Washington University in St. Louis - Department of Molecular Microbiology ( email )

One Brookings Drive
Campus Box 1208
Saint Louis, MO 63130-4899
United States

Mark S. Kuhlenschmidt

University of Illinois at Urbana-Champaign - Department of Pathobiology

601 E John St
Champaign, IL 61820
United States

Theresa B. Kuhlenschmidt

University of Illinois at Urbana-Champaign - Department of Pathobiology

601 E John St
Champaign, IL 61820
United States

William H. Witola

University of Illinois at Urbana-Champaign - Department of Pathobiology

601 E John St
Champaign, IL 61820
United States

Thaddeus S. Stappenbeck

Washington University in St. Louis - Department of Pathology and Immunology

One Brookings Drive
Campus Box 1208
Saint Louis, MO 63130-4899
United States

L. David Sibley

Washington University in St. Louis - Department of Molecular Microbiology ( email )

One Brookings Drive
Campus Box 1208
Saint Louis, MO 63130-4899
United States

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