A Stem-Cell-Derived Platform Enables Complete Cryptosporidium Development In Vitro and Genetic Tractability

Abstract

Summary 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 experimental 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 completion of the parasite life cycle in vitro . ALI cultures support parasite expansion > 100-fold and generate viable oocysts that are transmissible in vitro and to mice, causing infection and animal death. Transgenic parasite lines created using CRISPR/Cas9 were used to complete a genetic cross in vitro , demonstrating Mendelian segregation of chromosomes during meiosis. ALI culture provides an accessible model that will enable innovative studies into Cryptosporidium biology and host interactions.

A One Health Approach to Tackle Cryptosporidiosis

“…Recently, there has been some exciting progress on the in vitro culture of Cryptosporidium parasites using hollow-fibre technology [71] and the demonstration of the complete life cycle of C. parvum when cultured in human and mouse intestinal organoids [72,73]. Heo et al microinjected Cryptosporidium into the lumen of three-dimensional human-derived organoids [72], while Wilke et al infected monolayers of mouse-derived organoids cultured at an air-liquid interface in transwells [73]. Both culture systems support long-term growth (N20 days) and produce viable, infectious oocysts.…”

“…Both culture systems support long-term growth (N20 days) and produce viable, infectious oocysts. Additionally, Wilke et al generated transgenic parasites in vitro in organoid co-culture and used these parasites to perform a genetic cross [73]. These advances will help to reduce the numbers of animals used in research and for production of oocysts.…”

Defining Stage-Specific Activity of Potent New Inhibitors of Cryptosporidium parvum Growth In Vitro

“…However, this system requires microinjection of parasites and does not allow ready access for experimental manipulation. As an alternative system for long-term propagation of C. parvum, we have recently described a mouse enterocyte model that is based on the propagation of intestinal stem cells, followed by differentiation on two-dimensional (2D) transwell filters (22,23). Removal of the liquid medium from the upper chamber to create an air-liquid interface (ALI) induces differentiation of intestinal cell lineages and favors the growth of C. parvum (22,23).…”

Organoids – New Models for Host–Helminth Interactions

“…In particular, Toxoplasma gondii infects bovine and porcine small intestinal organoids [31]. Furthermore, the entire life cycle of Cryptosporidium parvum can now be modelled in murine and human small intestinal [33,34] and lung organoids [34]. Lung organoids have successfully recapitulated respiratory tract infections with C. parvum occurring in immune-competent and -deficient individuals [34].…”

“…For pathogens other than helminths, there are no reports on the impact of the dimensional conformation and architecture of organoids on the interactions with the host cells. Indeed, the life cycle of the protozoan parasite C. parvum can be replicated both in 3D and 2D organoids [33,34].…”