Cultivation, cryopreservation, and transcriptomic studies of host-adapted Cryptosporidium parvum and Cryptosporidium hominis using enteroids.

Cryptosporidium hominis and Cryptosporidium parvum are major causes of severe diarrhea. Comparative studies of them are hampered by the lack of effective cultivation and cryopreservation methods, especially for C. hominis . Here, we describe adapted murine enteroids for the cultivation and complete development of host-adapted C. parvum and C. hominis subtypes, producing oocysts infectious to mice. Using the system, we developed a cryopreservation method for Cryptosporidium isolates. In comparative RNA-seq analyses of C. hominis cultures, the enteroid system generated significantly more host and pathogen responses than the conventional HCT-8 cell system. In particular, the infection was shown to upregulate PI3K-Akt, Ras, TNF, NF-κB, IL-17, MAPK, and innate immunity signaling pathways and downregulate host cell metabolism, and had significantly higher expression of parasite genes involved in oocyst formation. Therefore, the enteroid system provides a valuable tool for comparative studies of the biology of divergent Cryptosporidium species and isolates.