Conditional cell reprogramming for modeling host‐virus interactions and human viral diseases

Abstract Conventional cancer and transformed cell lines are widely used in cancer biology and other fields within biology. These cells usually have abnormalities from the original tumor itself, but may also develop abnormalities due to genetic manipulation, or genetic and epigenetic changes during long‐term passages. Primary cultures may maintain lineage functions as the original tissue types, yet they have a very limited life span or population doubling time because of the nature of cellular senescence. Primary cultures usually have very low yields, and the high variability from any original tissue specimens, largely limiting their applications in research. Animal models are often used for studies of virus infections, disease modeling, development of antiviral drugs, and vaccines. Human viruses often need a series of passages in vivo to adapt to the host environment because of variable receptors on the cell surface and may have intracellular restrictions from the cell types or host species. Here, we describe a long‐term cell culture system, conditionally reprogrammed cells (CRCs), and its applications in modeling human viral diseases and drug discovery. Using feeder layer coculture in presence of Y‐27632 (conditional reprogramming, CR), CRCs can be obtained and rapidly propagated from surgical specimens, core or needle biopsies, and other minimally invasive or noninvasive specimens, for example, nasal cavity brushing. CRCs preserve their lineage functions and provide biologically relevant and physiological conditions, which are suitable for studies of viral entry and replication, innate immune responses of host cells, and discovery of antiviral drugs. In this review, we summarize the applications of CR technology in modeling host‐virus interactions and human viral diseases including severe acute respiratory syndrome coronavirus‐2 and coronavirus disease‐2019, and antiviral discovery.

cell senescence, and they also have very low yields and high variability from any original tissue specimens. As PDCM described above, organoids, iPSCs, and CRCs have been used for the generation or expansion of human normal cells, and these serve as great resources for cell biology and modeling human diseases including viral infections and antiviral discovery. 1,2 We summarize the characteristics of these approaches in Table 1. In this review, we focus on the applications of CRCs in modeling host-virus interactions and human viral diseases, and antiviral discovery.

| CR is rapid
We initially discovered that combination of feeder layers and a Rho kinase inhibitor, Y-27632, allows the generation of long-term cultures of both normal and tumor cells from keratinocyte and non-keratinocytes tissues. [3][4][5][6] The culture condition may convert or reprogram the whole-cell populations in synthetic medium to a stem cell-like status within 2 days, rather than a long-term clonal selection. 3,4 These Reprogrammed cells quickly stop proliferating or differentiate after removal of one of the culture conditions, either Y-27632 or feeder layer. Usually typical epithelial cell colonies are surrounded by feeder cells that can be visualized within 18 to 36 hours after initial plating from single-cell suspension. Thus, we termed this cell technology as "conditional reprogramming (CR)," and the resulting cells as "conditionally reprogrammed cells (CRC)," respectively. 1,3-6 As normal CRCs maintain their lineages and differentiation functions under in vitro three-dimensional (3D) or in vivo conditions, the CR technology has been widely used in basic and translational cancer biology, disease modeling, tissue regeneration, evaluation of drug toxicity, virus infections, and so on. Indeed, organoids 7-11 and CR technologies have been both recognized as the key new technologies by NIH precision oncology, 12,13 and have also been used

| CR technology is simple and cheap
Originally, the CR used irradiated mouse fibroblast cells (swiss mouse 3T3, J2 clone) and the Rho-associated kinase inhibitor (Y-27632) to propagate epithelial cells. 3,5,6 A few improvements have been used to simplify protocols using J2 conditioned medium, 3,5,6 hypoxia condition (1-2% O 2 ), 60 and combination with mTOR or TGF-beta, and SMAD inhibition [61][62][63] in the presence of Y-27632. CR technology is simple and cheap as there is no need for expensive reagents as matrigel for organoids, and robust as 1 × 10 6 cells can be generated from a needle biopsy within 7 days, and rapid as the whole populations of cells can be reprogrammed within 2 days instead of needing clonal selection. Figure 1 shows a diagram of normal cell cultures in CR conditions for long-term cultures and 3D (ALI, air-liquid interface; LLI, liquid-liquid interface; and organoids) conditions for ex vivo models of airway epithelial cells.

| CRCs MAINTAIN THEIR LINEAGE FUNCTIONS
As shown in Figure

| Human parvovirus
Human bocavirus 1 (HBoV1) often infects children and causes acute respiratory tract illness, such as pneumonia, induces, asthma exacerbations, and/or bronchiolitis, and some are life-threatening. 102 It has been shown that HBoV1 may infect well-differentiated or polarized human primary airway epithelium cultured at ALI cultures. 103  in HBoV1 genome amplification in ALI system. This is the first report to show that parvovirus DNA replicates in non-dividing cells autonomously. 104 Then, they also discovered that HBoV1 infection activates antiapoptotic proteins, thereby suppressing apoptosis but promoting pyroptosis. 105 Thus, CR-coupled ALI system may serve as a physiological model for study interactions of HBoV1 and host cells, and a system for antiviral discovery as well.

| Distal airway epithelial cells and influenza virus
Imai-Matsushima et al 109

| Hepatocyte cultures and liver diseases
Primary liver cancer is the 6th most frequent cancer type globally with high mortality, partially due to the lack of effective therapeutic options. The leading cause of liver cancer is cirrhosis due to viral hepatitis (HBV and HCV), aflatoxin, nonalcoholic fatty liver disease, or alcohol. The most common type is hepatocellular carcinoma  These suggest that CR technology can be used for expansion of normal and tumor cells in GU system. These cells may serve an ex vivo models for studies of viral infections in genital-urology, for example, SASR-CoV-2 induced injury of the kidney.
COVID-19 patients at the early stage of or asymptomatic or mild symptomatic patients usually have rapid replication of viruses at upper airway. Whether or not SARS-CoV-2 spreads to lower airway track is due to virus-host interaction, innate response, and local immune response. Severe COVID-19 patients with multiorgan injury are usually due less to virus replication-based direct injury and more to immunopathogenic injuries ( Figure 3).
SARS-CoV-2 and SARS-CoV share the same functional host-cell receptor ACE2, [130][131][132][133][134] and that SARS-CoV-2 possesses crucial amino acid residues for ACE2 binding. 133 ACE2 predominantly expresses in vascular endothelial cells, kidney and heart tissues, small intestine, and testes. 135 Recently, two reports demonstrated large amounts of SARS-CoV-2 in the upper airway and saliva. 116,123 Human-related physiological models are urgently needed for these studies of body site-specific or tissue-specific viral replication, innate immune response, and infectivity. As a functional and biological system, CR coupled with ALI/LLI culture will facilitate these studies and development of novel therapeutics ( Figure 1). Indeed, early study on SARS-CoV indicates that host cell differentiation or polarized epithelium and expression of ACE2 are both important for the susceptibility of human airway epithelia to SARS-CoV viral infection. 136 Indeed, Baric lab at UNC has used for ALI cultures of human airway epithelial cells (HAEs) for functional drug screening of SARS-CoV and SARS-CoV-2. 137,138 To overcome the difficulties with stable source and expense of primary human normal cells and variability of donors, CRCs from airway, GI, F I G U R E 3 Proposed diagram of SARS-CoV-2 replication and immunopathogenic injuries for COVID-19 patients. COVID-19, coronavirus disease-2019; SARS-CoV-2, severe acute respiratory syndrome coronavirus-2 GU tracts in combination with ALI or LLI will be a better choice for physiological systems for SARS-CoV-2 studies (Figure 1).

| SUMMARY
Owing to the limitations of current cell line and animal models, there is an urgent need for human physiological cell models for the study of viral infections and discovery of antiviral drugs. Here, we summarized the long-term cultures for human normal epithelial cells from respiratory tract, GI, and GU tracts using CR technology. Their cultures provide a stable source for normal cells from individuals and populations (race, age, gender, geography, etc.); CRC-coupled ALI/LLI/Organoids technologies (Figure 1) may serve as ex vivo physiological models for host-virus interactions and human viral disease, especially emerging and re-emerging virus infections. These will facilitate studies of virus entry, innate immune responses, viral replications, and drug discovery.