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

6

Infectious Diseases Division, Dept Internal Medicine1, Dept Surgery2, University of 7

Texas Medical Branch, 301 University Boulevard, Galveston TX 77555-0435 8

9

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

19

Running title: Cryptosporidium infects human primary cells. 20

21

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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24

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

44

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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

75

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

116

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

133

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

145

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

170

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

185

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

200

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

209

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

218

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

247

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

292

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

323

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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