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transcript
Human primary intestinal epithelial cells as an improved in vitro model for 1
Cryptosporidium infection 2
3
Alejandro Castellanos-Gonzalez1#, Miguel Cabada1, Joan Nichols1, Guillermo Gomez 2, 4
A, Clinton White Jr 1. 5
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Infectious Diseases Division, Dept Internal Medicine1, Dept Surgery2, University of 7
Texas Medical Branch, 301 University Boulevard, Galveston TX 77555-0435 8
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10
# Address correspondence to: 11
Alejandro Castellanos-Gonzalez 12
Infectious Disease Division, Department of Internal Medicine 13
University of Texas Medical Branch 14
301 University Boulevard 15
Galveston, TX 77555-0435. USA 16
Tel 409-772-3729 17
Email: alcastel@utmb.edu 18
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Running title: Cryptosporidium infects human primary cells. 20
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Key words: Cryptosporidium, human primary cells, infection model. 22
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Copyright © 2013, American Society for Microbiology. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.01131-12 IAI Accepts, published online ahead of print on 18 March 2013
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Abstract 25
The study of human intestinal pathogens has been limited by the lack of methods for 26
long-term culture of primary human intestinal epithelial cells (PECs). The development 27
of infection models with PECs would allow a better understanding of host-parasite 28
interactions. The objective of this study was to develop a novel method for prolonged In 29
vitro cultivation of PECs that can be used to study Cryptosporidium infection. We 30
isolated intact crypts from human intestine removed during weight loss surgery. The 31
fragments of intestinal layers were cultivated with culture medium supplemented with 32
growth factors and anti-apoptotic molecules. After 7 days, the PECs formed self-33
regenerating cell clusters forming villi that resemble intestinal epithelium. The PECs 34
proliferated and remained viable for at least 60 days. The cells expressed markers for 35
intestinal stem cells, epithelial cells, and mature enterocytes. PECs were infected with 36
Cryptosporidium. In contrast to older models in which parasite numbers decay, the 37
burden of parasites increased for over 120 hours. In summary, we have described a 38
novel method for cultivation of a self-regenerating human epithelial cells from small 39
intestinal crypts, which contains both intestinal stem cells and mature villus cells. We 40
presented data that suggest these cells support Cryptosporidium better than existing 41
cells lines. Thus, PECs should provide an improved tool to study host-parasite 42
interactions for Cryptosporidium and other intestinal pathogens. 43
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Introduction 46
The intestinal epithelium provides a barrier to invasive pathogens, but is also the major 47
target for a number of intestinal pathogens, including rotavirus, norovirus, 48
Cryptosporidium, Giardia, Salmonella and some strains of E. coli (1-4). Together, these 49
pathogens are major causes of acute and persistent watery diarrhea, which remains a 50
major cause of morbidity and mortality worldwide (5). The understanding the interaction 51
between epithelial cells and parasites would facilitate the identification of targets or 52
drugs needed to treat them. However, progress in research on these organisms has 53
been hampered by limitations of current experimental models (6). Because animal 54
models are suboptimal for some human infections, human colonic cell lines have been 55
used as an alternative to study intestinal pathogens (7, 8). The utility of cell lines is 56
limited by the fact they are not derived from small intestine and are transformed. In 57
addition, they do not readily support some of the major pathogens e.g. Cryptosporidium 58
spp., which undergoes incomplete replication, and norovirus, which has not been 59
propagated in vitro (9-11). The culture of primary human intestinal epithelial cells 60
(PECs) that retain human characteristics would be an alternative to study human 61
intestinal infections. Short term cultured PECs have been used to study 62
cryptosporidiosis (12). However, cultured PECs are short-lived cells and undergo 63
apoptosis when cultured in vitro (13). Recent studies have shown the feasibility of long 64
term culture of stem cells obtained from adult human intestines, stem cells can be 65
expanded in basement membrane matrix as matrigel and cultured for long-term (14, 66
15). However, although these models form villi, the matrigel is not optimal for 67
Cryptosporidium infection since the pore size of this matrix limits the access of the 68
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sporozoites to target cells. We hypothesized that using intact crypts cultured in medium 69
but supplemented with growth and differentiation factors with antiapoptotic molecules 70
would allow the establishment a system for long-term cultures of PECs that support 71
infection of intestinal pathogens. In the present work, we showed the feasibility of long 72
term cultured PECs that can be infected with Cryptosporidium which provides an 73
improved method for in vitro cultivation of the organism. 74
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Materials and Methods 76
PECs culture media. Wash medium consisted of D-MEM/F-12 (Dulbecco's Modified 77
Eagle Medium:Nutrient Mixture F-12) medium supplemented with 5% fetal bovine 78
serum (FBS), and antibiotic-antimycotic (1x) solution [Life technologies, Carlsbad, CA]. 79
Transport medium consisted of wash medium supplemented with recombinant 80
osteoprotegerin ([100 ng/ml], R&D systems, Minneapolis MN). Maintenance medium 81
was transport medium supplemented with epithelial growth factor ([50 ng/ml] Life 82
technologies, Carlsbad, CA), Noggin ([100 ng/ml] R&D systems, Minneapolis MN ), R-83
spondin ([1 µg/ml] R&D systems, Minneapolis MN ) and osteoprotegerin ([100 ng/ml], 84
R&D systems, Minneapolis MN). Differentiation medium consisted of enterocyte 85
differentiation medium containing D-MEM plus butyrate (Becton and Dickinson, Franklin 86
Lakes, NJ) supplemented with epithelial growth factor, noggin and R-spondin. 87
88
Isolation and culture of PECs. Jejunal tissues were removed during gastric bypass 89
surgery performed for weight reduction. Jejunal tissues that would otherwise have been 90
discarded were obtained with IRB approval. In the operating suite tissue was placed in a 91
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50 ml conical tube containing 25 ml of transport medium and then transported 92
immediately to the laboratory on ice. Within 3 hr, the tissue was washed inverting 93
gently the tube to decant the supernatant and adding washing medium (three times). 94
The tissue was dissected with a scalpel and the mucosal surface was exposed, and the 95
superficial mucus was removed mechanically with a cell scraper (Corning life science, 96
Manassas, VA). The mucosal surface was covered with 5 mM EDTA-PBS 1X for 3 min 97
at room temperature and sheets of epithelial cells including intact crypts were removed 98
mechanically with a scalpel. After centrifugation (40 x g, 10 min), the supernatant 99
containing isolated cells was discarded. The pellet was re-suspended in 5 ml of red 100
blood cells lysis solution (8.3 g/L ammonium chloride in 0.01 M Tris-HCl, 10 min at room 101
temperature), after incubation 20 ml of washing medium was added to dilute the lysis 102
buffer. The sample was centrifuged (40 x g, 10 min) and then the supernatant was 103
discarded. The pellet was re-suspended in 20 ml of washing medium and centrifuged 104
(40 x g, 10 min) three times, the sheets containing epithelial cells and crypts were gently 105
scraped from the bottom and aspirated with a micropipette tip (size 1ml) with the end of 106
the tip previously cut with a scalpel; cells were gently re-suspended in 5 ml maintenance 107
medium and plated in 12 well culture plates in 250 µl/well (37⁰C, 5% CO2, 7 d). The 108
maintaining medium was carefully replaced daily. After 7d, the cells were harvested by 109
aspiration and centrifugation (40 x g, 10 min). The supernatant containing dead cells 110
was discarded and the epithelial cell layers were concentrated and plated in 96 well 111
round bottom plates (~100 fragments in 50 µl of maintenance medium per well, 37⁰C, 112
5% CO2. Additional maintenance medium (25 μl) was added every other day up to 60 d. 113
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For the experiments of characterization maintenance medium was replaced with 25 µl of 114
differentiation medium 48 h before the cells were harvested for characterization. 115
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Viability assays, histology and electron microscopy. Viability of the PECs was 117
assessed by a two color fluorescence cell viability assay by using live/dead viability kit 118
(Life technologies, Carlsbad, CA) to detect calcein (live cells) and ethidium homodimer 119
(dead cells) by fluorescent microscopy at 495 nm. For histological analysis, PECs were 120
fixed in 10% formalin and embedded in paraffin wax, for infection experiments PECs 121
were challenged sporozoites of C. parvum and then the infected PECs were fixed as 122
before. Sections were stained with methylene blue, hematoxylin-eosin or examined by 123
fluorescence. For transmission electron microscopy, the PECs were fixed (2.5% 124
paraformaldehyde and 0.1% glutaraldehyde in 0.05 M cacodylate buffer, pH 7.3, with 125
0.03% trinitrophenol and 0.03% CaCl2), washed (0.1 M cacodylate buffer), scraped off 126
the flasks and pelleted. The pellets were post-fixed (1% OsO4 in 0.1 M cacodylate 127
buffer), stained (1% uranyl acetate in 0.1 M maleate buffer), dehydrated in ethanol and 128
embedded (Poly/Bed 812, Polysciences, Warrington, PA). Ultrathin sections were cut 129
(Reichert-Leica Ultracut S ultramicrotome), stained with lead citrate and examined by 130
transmission electron microscopy using a Philips 201 or CM-100 electron microscope at 131
60 kV. 132
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Immunofluorescence and ELISA assay. The PECs were characterized by 134
immunofluorescence using fluorescein isothiocyanate (FITC) anti-pan-cytokeratin as a 135
marker of epithelial cells (Sigma-Aldrich, St. Louis, MO) and FITC-anti-vimentin as a 136
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marker for fibroblasts (Sigma-Aldrich, St. Louis, MO). For parasite detection we used 137
the Cryptosporidium parvum gp900 and gp40 Antibody (GenWay Biotech, San Diego 138
CA) at working dilution 1:50 and Phalloidin Alexa fluor 568 (Life technologies, Grand 139
Island, NY) at concentration 1:1000. Cell proliferation was evaluated using Alexa Fluor 140
647-mouse anti-human Ki-67 (BD Biosciences, San Diego, CA). The production of 141
alkaline phosphatase was measured in the supernatants of cells from days 30-35 in 142
culture using the SensoLyte pNPP alkaline phosphatase ELISA Kit following the 143
instructions of the vendor (AnaSpec, Fremont, CA). 144
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RNA extraction and PCR assays. PECs were harvested by centrifugation (40 x g x 10 146
min) and the supernatant was discarded, then the pellets were stored frozen (-20 C°). 147
The frozen cells were suspended in guanidine buffer and disrupted with QIAshredder 148
columns (Qiagen, Valencia, CA). RNA was isolated using the RNAeasy KIT Plus 149
(Qiagen, Valencia, CA). The purity and concentration were analyzed by 150
spectrophotometry (Nanodrop 1000, Thermo Scientific, Pittsburgh, PA). The presence 151
of RNA of epithelial cell markers was analyzed using the one step real time reverse 152
transcription-PCR Sybr green kit (Life technologies, Carlsbad, CA). Epithelial cell RNA 153
(25 ng) was extracted from 30 days old PECs, freshly isolated intestinal cells, HCT-8 154
cells and fibroblasts were analyzed using previously described primers for cytokeratin 8, 155
vimentin, aldolase, mucin, lysozyme, LgR5, Ki67 and human 18s rRNA (16-18). 156
Following reverse transcription (50ºC, 15 min), denaturing (95ºC 5 min), and 40 cycles 157
melting (95ºC 30s) and annealing-extension (60ºC 1 min), the amplicons were detected 158
by electrophoresis in agarose gels at 1% (Fig. 2). For infection experiments, the 159
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parasite burden was quantified by RT-PCR from infected PECs obtained at different 160
time points (see below). For RT-PCR amplification we used 100 ng of RNA as template 161
and we used the method and the specific primers previously reported to detect 18S 162
rRNA (19). To analyze the number of parasites we used the ABI prism software using 163
as reference the ct values obtained from an standard curve prepared with known 164
numbers C. parvum oocysts ranging from 1x102 to 1x106 organisms. To compare 165
infection efficiency HCT-8 cells were infected as before and the numbers of parasites 166
were determined as mentioned above. Heat killed (95ºC) Cryptosporidium sporozoites 167
were used as negative controls and human 18S rRNA was used as reference gene to 168
normalize the samples. 169
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Cryptosporidium parvum infection. Cryptosporidium parvum oocysts Iowa strain 171
(Sterling Parasitology Laboratory, University of Arizona, Tucson, AZ) were stained with 172
carboxyfluorescein diacetate succinamidyl ester (CFSE, CellTrace™ CFSE Cell 173
Proliferation kit, Life technologies, Carlsbad, CA ) and excysted using acidified water 174
and bile salts (7). After removing intact oocysts by centrifugation (500 g, 3 min), 175
sporozoites were suspended in culture media and filtered through a 3 microns filter 176
(Millipore, Billerica MA). No oocysts were seen in the filtered medium by microscopy. 177
Sporozoites (2x104) were added to each well containing PECs and incubated (5% CO2, 178
37⁰C), the supernatant was removed after 3 hr of infection and then fresh infection 179
medium (maintaining medium supplemented with 0.8% taurocholate) was added daily 180
up to harvesting. For fluorescent microscopy, unfixed cells from the supernatant were 181
analyzed 120 hr after the infection. Parasite burden was assessed by RT-PCR as we 182
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described before using total RNA extracted from infected PECs (including supernatant) 183
at 0, 24, 48, 72, 96 and 120 hrs after infection. 184
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Results 186
PECs remain viable up to 60 days and shows epithelial structure. We were able to 187
detect viable PECs from surgical specimens in the vast majority of cases (replicated 188
over 15 times). During the beginning of cell isolation a large number of dead cells and 189
small PECs fragments were found in the supernatant but these were discarded during 190
subsequent washing steps and then after 1 week of culture the layers containing PECs 191
were concentrated in 96 well plates and viability was monitored. After 2 months of 192
culture the viability assay carried out with attached, unfixed PECs showed that the cells 193
all stained with the vital dye (Fig. 1A), which indicates that the cells remained viable for 194
at least 60 days. By contrast, heat killed cells did not stain with the vital dye, but stained 195
with the dye for dead cells (Fig. 1B). Histological sections of the cultured PECs 196
confirmed the presence of crypts and villi (Fig. 1C) with typical enterocytes and goblet 197
cells (Fig. 1D) which suggested that PECs contained a mixed population of epithelial 198
cells. 199
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Cultured PECs shows epithelial cell characteristics. To confirm that the PECs were 201
in fact epithelial cells, we stained the cultured PECs with fluorescent antibodies to 202
cytokeratin and vimentin. Nearly all of the cells stained for cytokeratin, a marker for 203
epithelial cells (Fig. 2A top) but not for vimentin, a marker for fibrobasts (Fig. 2A 204
bottom). As shown in 2B cytokeratin expression persisted at similar levels throughout 205
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the time in culture, which indicates that the daughter cells produced during cultivation 206
maintain an epithelial phenotype (Fig. 2B). Levels of cytokeratin were similar to those 207
observed in fresh PECs and HCT-8 cells. 208
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Cultured PECs shows proliferation and differentiation markers. By electron 210
microscopy, we showed that the cultured PECs expressed both tight junctions and 211
microvilli (Fig. 3A). Within the cultured PECs some cells express the proliferation marker 212
Ki67 (Fig. 3B). We detected markers of stem cells (LGR5) and proliferating cells (Ki67) 213
by PCR (Fig. 3C). We also detected markers for differentiated villus cells including 214
aldolase, mucin, and lysozyme (Fig. 3C). The supernatants of 30 d old PECs contained 215
alkaline phospatase as is expected of mature epithelial cells (Fig. 3D). Thus the cultured 216
cells express markers for both long-lived stem cells as well as differentiated cells. 217
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Cryptosporidium parvum infection. We challenged the PECs with Cryptosporidium 219
sporozoites previously stained with CFSE with the aim to track the formation of later 220
stages, and as showed in figure 4A no oocyst like structures were observed after 221
filtration and before the inoculation. As expected because the low number of the initial 222
inoculum, we observed only very few intracellular forms during the first days of infection 223
(4H), however as is observed in 4B we showed that the supernatant obtained after 5 224
days of infection contained a large number of parasites positively stained for CFSE (Fig. 225
4B). Since sporozoites die few hours after the ecxystation, thus we concluded that 226
detection of stained intracelular forms and stained parasites in the supernatant should 227
be the result of parasite proliferation. Microscopic analysis of the forms found in the 228
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supernatant revealed the different morfology among stained forms, we detected 229
parasites with shapes and sizes similar to those found in sporozoites and merozoites 230
(Fig. 4C), in addition we detected uninucleated parasites with caracteristics similar than 231
trophozoites (Fig. 4D), we oberved parasites multinucleated with a crescent shape wich 232
could correspond to a meronte like structure (Fig. 4D), Oocysts, the infective product of 233
sexual reproductive, also were noted in the supernatants (4F). Since Cryptosporidium 234
RNA is stable just for few hours after parasites die, this has been used to determine 235
parasite viability (20), then increases in RNA level over the time should be the result of 236
parasite proliferation. Thus to determine the growth characteristics of the parasites, we 237
compared parasite burden by quantitative reverse transcriptase real time PCR for 238
parasite RNA. Infection of HCT-8 cells (the cell line said to best support 239
Cryptosporidium) resulted in a gradual decay of the amount of parasite RNA. By 240
contrast, parasite RNA increased after infection of primary epithelial cells for periods of 241
at least 120 hrs (Fig. 4G). Immunofluorecence (IF) staining with anti-crypto antibody 242
showed the presence of intracelular forms in sections of PECs cultured for 120 hrs (Fig. 243
4H) and confirmed the presence of Cryptosporidium in cultured PECs, the parasite 244
staining was similar than the observed in previous reports of HTC-8 cells infected with 245
Cryptosporidium (21). 246
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Discussion 248
The first objective in this study was to develop a system to establish a long-term culture 249
of PECs which retains the phenotypic properties of an epithelial layer including 250
progenitor cells, proliferative cells, differentiated cells, and senescent cells. A 251
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substantial obstacle in the cultivation of intestinal epithelial cells is the onset of 252
apoptosis (anoikis) when epithelial cells are detached from the basement membrane 253
(22). Previous studies have showed the utility of anti-apoptotic molecules to extend the 254
life of intestinal primary cells after detaching them from the intestinal tissue (14, 23). 255
Thus here we added recombinant osteoprotegerin (OPG) during the transport of the 256
tissue and immediately after PECs isolation to prevent anoikis, OPG is a TNF receptor 257
family member, which acts as decoy by binding TRAIL and TRANCE blunting the 258
apoptosis pathway (24-26). We previously demonstrated that intestinal epithelial cells 259
produce OPG in response to Cryptosporidium infection (25). We reasoned that OPG 260
might improve survival of the cells especially during first 48 hrs of culture. Although we 261
observed high rate of death cells on days 1 to 3, over the time we observed that after 262
OPG addition a large number of PECs survived beyond several days of culture. Thus 263
the dying cells observed on first days are likely terminal differentiated enterocytes. On 264
the other hand undifferentiated or cells in early process of differentiation survived. Thus, 265
OPG might be critical a molecule to consider for preventing apoptosis in the beginning 266
of long term PECs cultures. Despite addition of antiapoptotic factors, normal epithelial 267
cells turn over every 7 days. Thus, the presence of viable cells observed in prolonged 268
culture (after 1 week) is likely related to proliferation rather than with the extension of 269
the life span of partially differentiated cells due OPG. During the isolation of PECs, we 270
isolated intact crypts. We hypothesized that the cultured clusters of PECs should 271
contain mixed population of epithelial cells including stem cells. Intestinal stem cells can 272
be expanded in vitro when cultured in the presence of Wnt pathway activators as noggin 273
and R-spondin (14, 23, 27, 28). Therefore here we added recombinant noggin proteins 274
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to induce proliferation in the cultured PECs. We confirmed the presence of stem cells in 275
long term cultured PECs by staining and PCR for LGR-5 and Ki-67. At difference of 276
cancer cell lines the Intestinal stem cells have the capacity to produce all 4 types of 277
intestinal cells (29). Here we hypothesized that differentiation factors could be used to 278
direct and control differentiation of expanded populations of stem cells presents in the 279
culture. We used medium containing butyrate to induce differentiation. Butyrate is a 280
short-chain fatty acid normally produced by commensal bacteria that enhances 281
functional differentiation of enterocytes (30) via the PTEN/phosphoinositide 3-kinase 282
pathway (31). When cultured with butyrate, we noted the presence of live cells with 283
crypt and villus structures, which expressed markers of differentiated cells including 284
microvilli, tight junctions, and alkaline phosphatase production. The histology showed 285
the presence of well differentiated enterocytes as well as goblet cells. This contrasts 286
with studies of epithelial cells differentiated from stems cells. In the latter case, cells 287
grow in organoids (cyst like structures). The latter have some markers of mature cells, 288
but do not form complete villus structures. Thus, here we demonstrated that PEC can 289
be successfully propagated in vitro for a period of at least 60 days and as well as 290
producing markers of mature villi. 291
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The second objective in this work was to demonstrate the feasibility of infecting long 293
term cultured PECs with the intestinal parasite Cryptosporidium. PECs have a number 294
of advantages over cancer cell lines since they may more accurately reflect in vivo 295
conditions than immortalized cells. In particular, primary cells allow direct and 296
meaningful examination ex vivo of species tropism and the importance of specific-297
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receptor ligand interaction. In previous studies we have used human intestinal tissues 298
as explants as an alternative to study Cryptosporidium infection (25, 32). However we 299
have observed a low rate of infection even when large numbers of parasites are used 300
and also we have observed a high rate of apoptosis even in uninfected cells after 48 hrs 301
of cultivation. Here we have hypothesized that improvement in PECs culture developed 302
in our laboratory could led in an improvement of cryptosporidiosis model. To 303
demonstrate that PECs support Cryptospordium infection, we challenged the PECs with 304
low numbers of filtered sporozoites of C. parvum with the aim of detecting larger 305
amounts of parasites after culture. Since is difficult to characterize the infection by EM 306
because the low number of infected cells (due the inoculums) especially during first 307
days of infection, we used more sensitive methods as RT-PCR to track the infection 308
over the time (Fig. 4G). In contrast to cell lines, we noted proliferation as demonstrated 309
by drastically increasing parasite RNA for over 120 hrs. The quantity of parasite mRNA 310
was twice as high as in HCT-8 cells, the cells line which best supports Cryptosporidium 311
in vitro (33). In addition, at difference of previous models here we present evidence that 312
Cryptosporidium is completing its life cycle, this evidence is based in the fact that the 313
infection is started just with sporozoites (pre-stained with a tracking dye) and then we 314
observed stained asexual forms as merozoites and trophozoites (probably detached 315
from death cells) and sexual forms including thin wall oocysts in the supernatant after 316
120 hrs of infection (Fig 4C-F). In conclusion we were able to produce a stable system 317
to culture PECs which are able to persist in culture for at least 60 days. It supports 318
Cryptosporidium better than previous in vitro models. Since the cultured PECs retained 319
intestinal characteristics we anticipated that method described here should provide an 320
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improved tool to study host-parasite interactions for Cryptosporidium and other intestinal 321
pathogens. 322
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324
Acknowledgements and support 325
We thank Mary K. Estes, PhD, and Don Powell, MD, for their valuable suggestions and 326
comments regarding the manuscript. This study was supported in part by the Institute 327
for translational Sciences (ITS) at the University of Texas Medical Branch at Galveston 328
“Supported in part by grant 1UL1RR029876-01 from the National Center for Research 329
Resources, National Institutes of Health. 330
331
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423 424 425 Figure 1. Viability and morphology of the cultured PEC. The viability of cultured 426
PEC was determined by fluorescent live/death assay (A), unfixed cells co-stained with 427
the vital dye calcein (green) and the marker of dead cells ethidium homodimer (red) at 428
488 nm (no dead cells are observed). As control heat killed cells are shown (B), scale 429
bars ~100 µm. Histological analysis of cultured cells (C), paraffin wax sections of the 430
PECs stained in methylene blue, villus (red arrows) and crypts (black arrows) are 431
shown, space corresponding to the mouth of the crypts is indicated (black asterisks), 432
scale bar ~50 µm. Morphology of the PEC (D), enterocytes with a typical cylinder shape 433
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(black) and goblet cells with the typical rounded shape (red) are marked in arrows, scale 434
bar ~30 µm. 435
436
Figure 2. Cytokeratin expression in cultured PECs by immunoflourescence and 437
RT-PCR. Expression of cytokeratin (Ctk) and vimentin (Vim) was analyzed by 438
Immunofluorescence (A). Cultured PECs stained with Anti-Cytokeratin-FITC (top row 439
left) or Anti-Vimenti FITC (bottom row left) were counterstained with DAPI (top and 440
bottom center), overlaid images are showed (top and bottom right), scale bar ~40 µm 441
(B). RT-PCR analysis in agarose gel 1% shown the expression of cytokeratin 8 (Ctk 8), 442
vimentin (Vim), and 18s rRNA (18s) through the time (C). 443
444
Figure 3. Markers of proliferation and differentiation in cultured PECs. Electron 445
micrograph of cultured cells (A) shows the presence of tight junctions (black arrow) and 446
microvilli (black asterisks). The nuclear proliferation marker Ki67 was detected (B) by IF 447
using a monoclonal AB anti-Ki67-PE (white arrows), cells were counterstained with 448
DAPI, scale bar ~10 µm. Differentiation and proliferation markers detected by PCR (C): 449
Aldolase (Aldo), Mucin (Muc), Lysozyme (Lys), Leucin rich receptor (LgR5), Ki67 and 450
18s rRNA (18s). Cultured PECs alkaline phospatase production. Activity of alkaline 451
phosphatase expressed in milliUnits from supernatants collected at day 0 (base line) or 452
day 5 from 30 days old cultured PECs (D). 453
454
Figure 4.- PECs infected with C. parvum. Sporozoites were filtered and stained with 455
CFSE before infection (A). After 5 days of infection the supernatant showed (objective 456
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40X) abundant stained parasites containing oocysts like structures (B). The 457
supernatant was analyzed in detail (objective 100X) showing the presence of: 458
merozoites (C), trophozoites (D), meronts (E) and mature oocyst (F). Scale bar = 10µm 459
in A and B and bar = 1 µm in C-F. Quantification of the number of parasites in infected 460
PECs by real time RT-PCR (G). Low inoculums of filtered sporozoites (1x102) were 461
added to 2 weeks old cultured PECs (red and blue) and HCT-8 cells (Black). The 462
number of parasites was evaluated up to 5 days. Red and blue lines are means of 2 463
independent experiments (SD of PCR triplicates are not observed in the figure), the total 464
number of parasites in the sample was determined using a standard curve with a known 465
number of parasites. Infection of PECs was confirmed by immunefluorescence by 466
detecting the monoclonal antibody anti Cryptosporidium labeled in green (H). 467
468
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