Investigation of murine host sex as a biological variable in epithelial barrier function and muscle contractility in human intestinal organoids

Abstract

Intestinal failure (IF) occurs when intestinal surface area or function is not sufficient to support digestion and nutrient absorption. Human intestinal organoid (HIO)-derived tissue-engineered intestine is a potential cure for IF. Research to date has demonstrated successful HIO transplantation (tHIO) into mice with significant in vivo maturation. An area lacking in the literature is exploration of murine host sex as a biological variable (SABV) in tHIO function. In this study, we investigate murine host SABV in tHIO epithelial barrier function and muscle contractility. HIOs were generated in vitro and transplanted into NSG male and female mice. tHIOs were harvested after 8–12 weeks in vivo. RT-qPCR and immunohistochemistry were conducted to compare tight junctions and contractility-related markers in tHIOs. An Ussing chamber and contractility apparatus were used to evaluate tHIO epithelial barrier and muscle contractile function, respectively. The expression and morphology of tight junction and contractility-related markers from tHIOs in male and female murine hosts is not significantly different. Epithelial barrier function as measured by transepithelial resistance, short circuit current, and FITC-dextran permeability is no different in tHIOs from male and female hosts, although these results may be limited by HIO epithelial immaturity and a short flux time. Muscle contractility as measured by total contractile activity, amplitude, frequency, and tension is not significantly different in tHIOs from male and female hosts. This data suggests that murine host sex may not be a significant biological variable influencing tHIO function, specifically epithelial barrier maintenance and muscle contractility, though limitations exist in our model.

Introduction

Intestinal failure (IF) is a condition where intestinal length, surface area, or function is not sufficient to support digestion and nutrient absorption (1). Short bowel syndrome is the major cause of intestinal failure and observed at a rate of 24.5 per 100,000 live births (2). Infants with IF often require parenteral nutrition (PN), and while the ultimate goal is to achieve enteral autonomy through intestinal adaptation, many patients remain PN dependent (1). Patients with prolonged PN dependence have increased morbidities such as central line associated bloodstream infection, mechanical central line complications, and intestinal-failure associated liver disease (1). Other medical and surgical treatment options such as teduglutide, bowel lengthening procedures and intestinal transplant while beneficial are not always curative, suggesting that there is significant room for improvement in management strategies for this patient population and a desperate need for better therapies (1). Human intestinal organoid (HIO)-derived tissue engineered intestine is a potential curative therapy to regenerate absent bowel and restore intestinal function in IF patients (3). However, current HIO models do not yet recapitulate the complexities of human intestinal function and are still under development.

Research to date has demonstrated successful transplantation of HIOs (tHIOs) grown in vitro into immunocompromised mice with significant in vivo maturation (4). Current investigations aim to improve the established HIO model by incorporating missing elements such as the enteric nervous system, immune system, and exposure to luminal contents such as nutrients and microbiota (5, 6). An additional area lacking in the present HIO literature is the exploration of murine host sex as a biological variable (SABV) in tHIO development and function. The majority of tHIO publications report utilizing only male murine hosts or simply do not report host sex (3, 4, 7–12). Only one previous study reported growing tHIOs in both male and female mice, but sex-related differences were not described (5). Historically, a majority of animal research has been conducted in male models, and Shansky suggests that biological sex is an avoided variable that could have significant influence on research outcomes across all fields (13).

Sex hormones include estrogens, androgens, and progestins and play an crucial role in the reproductive system, but also influence nonreproductive systems such as the immune, cardiovascular, and central nervous systems, and even the gastrointestinal tract (14). Prior studies have demonstrated that human hematopoietic stem cell engraftment is superior in female versus male nonobese diabetic, severe combined immunodeficiency, gamma chain deficient (NSG) mice (15, 16) and that estrogen is responsible for this sex-associated difference in engraftment (16). Importantly, circulating sex hormones may also affect intestinal functions such as epithelial barrier function and muscle contractility. Van der Giessen et al. found that estrogen and progesterone upregulated tight junction proteins claudin-1 (CLDN1), claudin-2 (CLDN2), occludin (OCLN), and zonula occludens-1 (ZO-1) resulting in improved epithelial barrier function in inflammatory bowel disease (IBD) patient-derived organoids and 2D cell lines (17). Collins et al. found that female mice displayed changes in in vivo intestinal permeability following ovariectomy compared to rodents undergoing a sham procedure, further implicating the role of estrogen in maintaining gut barrier function (18). Recently, in a mouse model of malnutrition, the malnourished young adult males, but not females, were found to have increased gut permeability and an exaggerated intestinal contractile response when compared to sex-matched controls (19). Additionally, in a study of enteric glial ablation, Rao et al. identified sex-dependent differences in glial regulation of intestinal motility in mice (20). It is established that the epithelium within tHIOs is influenced by circulating humoral factors (3). Watson et al. elegantly demonstrated that HIOs transplanted beneath the kidney capsule of murine hosts who had undergone ileocecal resection demonstrated the same signs of intestinal adaptation that the murine host intestine exhibited including significantly increased villus height, higher rate of crypt fission, and increased proliferative index within intestinal crypts (3). Thus, similarly, circulating sex hormones may also influence tHIO development and function.

These findings suggest that sex hormones may influence epithelial barrier function and muscle contractility in tHIOs. These potential sex-related differences will be critical to understand as we continue to optimize HIO-derived tissue-engineered intestine as a therapy for patients suffering from IF. In this study, we aim to investigate murine host SABV in epithelial barrier and muscle contractile function of tHIOs. Based on published data, we hypothesized that tHIOs grown in female hosts would display enhanced intestinal epithelial barrier function, whereas tHIOs from male hosts would demonstrate improved muscle contractility.

Materials & Methods

Animals

Animals were housed at The Center for Laboratory Animal Medicine and Care (CLAMC) at The University of Texas Health Science Center at Houston (UTHealth). Surgical procedures were performed with the approval of the Animal Welfare Committee (protocol #AWC-20–0044). The mouse colony was established from breeder nonobese diabetic, severe combined immunodeficiency, gamma chain deficient (NSG) mice from The Jackson Laboratory.

Generation of human intestinal organoids

Protocols related to the use of human embryonic stem cells (hESCs) were approved by the UTHealth Stem Cell Research Oversight Committee (protocol #SCRO-21–02). HIOs were generated as previously described using hESCs (H9, WiCell) through directed differentiation in vitro (4). Briefly, the hESCs were maintained in mTeSR Plus (STEMCELL technologies) media on hESC-qualified Matrigel (Corning). hESC media was replaced every second day. The hESCs were recovered using ReLeSR (STEMCELL technologies) upon reaching a confluence of 70%. The recovered hESCs were plated at a density of ~9500 cells per cm² for maintenance plates and ~40,000 cells per cm² for differentiation plates. When the monolayers on the differentiation plates reached a confluence of ~70–80%, the mTeSR Plus was replaced with RPMI 1640 (Gibco) supplemented with 100ng/mL Activin A (Cell Guidance Systems), NEAA 100X (Gibco), and daily increasing concentrations (0, 0.2, 2%) of defined FBS (HyClone). This media was changed daily for three days to induce differentiation into the definitive endoderm. Mid-hindgut spheroid formation was induced after 3 days with RPMI 1640 (Gibco) supplemented with 2% defined FBS (Hyclone), NEAA 100X (Gibco), 500ng/mL FGF4 (Peprotech) and 3μM Chiron 99021 (Tocris). This media was replaced daily for 4 days. Mid-hindgut spheroids were collected at day 4 and suspended in droplets of GFR Basement Membrane phenol-free Matrigel (Corning) to provide a 3D microenvironment that supports organoid growth. The spheroids were maintained in Advanced DMEM/F-12 (Gibco) supplemented with N2 (Gibco), B27 (Gibco), 10mM HEPES (Gibco), 2mM Glutamax (Gibco), 20% R-Spondin-1 conditioned media (Texas Medical Center Digestive Disease Center GEMS Core, Enteroid/Organoid Sub-core) and 100ng/mL EGF (ThermoFisher Scientific). Spheroids were also treated with 10% Noggin conditioned media (Texas Medical Center Digestive Disease Center GEMS Core, Enteroid/Organoid Sub-core) for the first three days. The media was changed every 3–4 days for up to 37 days. HIO in vitro growth was monitored on a Nikon Eclipse TS100 microscope (Nikon). Day 27–37 HIOs were transplanted into NSG mice.

Transplantation of human intestinal organoids

HIOs were transplanted under the left kidney capsule of adult age-matched 9–18 week old male (n=30) and female (n=30) NSG mice as described previously (4). Briefly, day 27–37 HIOs were released from Matrigel with ice-cold phosphate-buffered saline (PBS). A small incision was made on the left flank of the murine host to expose the kidney and a subcapsular pocket was generated. A single HIO was transplanted under the left kidney capsule. The muscle layer and skin was sutured closed and analgesia was provided for two days after the operation. The mice were humanely euthanized 8–12 weeks after transplantation surgery and the tHIO harvested. For the majority of tHIOs, one half of the tHIO was fixed in 4% paraformaldehyde (PFA) for tissue processing and histology and the other half of the tHIO was snap frozen for molecular analysis by RT-qPCR (male host n=17 and female host n=17). The next batch of tHIOs (male host n=6 and female host n=6) were allocated for analysis in the Ussing chamber, but 3 of the 12 tHIOs were not large enough for use in the chamber when harvested (male host n=2 and female host n=1). The remaining 9 tHIOs were adequate in size and the tHIOs were mounted in the Ussing chamber (male host n=4 and female host n=5). The final batch of tHIOs (male host n=7 and female host n=7) were allocated for analysis in the contractility apparatus, but 2 of the tHIOs were not large enough for use in the apparatus when harvested (female host n=2). The remaining 12 tHIOs were adequate in size and a strip from each tHIO was mounted in the contractility apparatus and after data recording was subsequently fixed in 4% PFA for tissue processing and histology while the remaining tHIO tissue was snap frozen for molecular analysis (male host n=7 and female host n=5).

Tissue Processing

tHIOs were placed in 4% PFA at room temperature (RT) for 1 hour. tHIOs were then washed in PBS three times for 5 min each. After washing, tHIOs were placed in 70% ethanol overnight at RT. tHIOs were subsequently dehydrated from 70% to 95% to 100% ethanol for an hour each, followed by xylene for an additional hour, and finally melted paraffin. tHIOs were embedded in paraffin blocks. Paraffin-blocked tHIOs were then sectioned at 10μm using a rotary microtome (LEICA RM 2155; Leica Microsystems, INC., Buffalo Grove, IL) and placed on glass slides (Surgipath) at RT. Paraffin-sections were stained with hematoxylin and eosin (H&E).

Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR)

tHIOs were homogenized with a Polytron PT3100 homogenizer (Kinematica). RNA was then extracted from the tHIOs using the E.Z.N.A Total RNA kit I (Omega Bio-Tek) according to the manufacturer’s instructions. Take3 Micro-Volume Plate (BioTek) in a Cytation 5 reader (BioTek) was used to determine RNA concentration and purity. The cDNA was synthesized using an iScript reverse transcription kit (BIO-RAD) with a Mastercycler (Eppendorf). RT-qPCR was performed using PowerUp SYBR Green Master Mix (ThermoFisher Scientific) according to the manufacturer’s instructions for the fast protocol on a CFX Touch Real-time PCR Detection System (BIO-RAD). Primers were obtained from Integrated DNA Technologies and are listed in Table 1.

Table 1 –

RT-qPCR primers

Gene Symbol	Gene name	Forward Primer	Reverse Primer
GAPDH	Glyceraldehyde 3-phosphate dehydrogenase	GAA GTT GAA GGT CGG AGT	TTG AGG TCA ATG AAG GGG TC
CLDN3	Claudin 3	AAC ACC ATT ATC CGG GAC TTC T	GCG GAG TAG ACG ACC TTG G
CLDN7	Claudin 7	ATC CCT ACC AAC ATT AAG TAT GAG TT G	TGC ACC TCC CAG GAT GAC TAG
CLDN15	Claudin 15	CTG CGC TGC ACC AAC ATT G	GGT ACA AGG GGT CGA AGA AGT
OCLN	Occludin	ACA AGC GGT TTT ATC CAG AGT C	GTC ATC CAC AGG CGA AGT TAA T
TJP1	Tight junction protein 1	ACC AGT AAG TCG TCC TGA TCC	TCG GCC AAA TCT CAC TCC
VIM	Vimentin	AGT CCA CTG AGT ACC GGA GAC	CAT TTC ACG CAT CTG GCG TTC
ACTA2	α smooth muscle actin	GTG TTG CCC CTG AAG AGC AT	GCT GGG ACA TTG AAA GTC TCA
KIT	KIT proto-oncogene receptor tyrosine kinase	CGT GGG CGA GAT TAG G	CTT TCC CAT ACA AGG AGC G

Immunohistochemistry (IHC) and microscopy

Paraffin-sections were rehydrated starting from Xylene, 100% ethanol, 95% ethanol, 70% ethanol, to deionized water for 3 minutes each, followed by permeabilization with 0.5% Triton X-100 (Sigma) for 20 minutes at RT. Antigen retrieval was performed using a steamer with 1X Citrate Buffer pH6.0 (Abcam) for 20 minutes. Slides were then cooled at RT for an additional 20 minutes, followed by cover-plating in sequenza chambers (Thermo Scientific) using 0.5% Triton X-100 for slides to adhere to the cover-plate. Slides were washed 6 times with PBS followed by blocking serum consisting of 5% Normal Donkey Serum (Jackson Immuno Research) in PBS with 0.05% Tween 20 (Sigma) for 1 hour at RT. Primary antibodies (Table 2) were diluted in blocking serum and incubated overnight at 4C. The next day, slides were washed 6 times in PBS followed by incubation in secondary antibody (Table 3) for 90 minutes at RT. Slides were then washed 6 times in PBS, removed from sequenza chambers and cover-slipped using Vectashield DAPI (Vector Laboratories) for imaging. Slides were imaged on a Nikon Eclipse Ti microscope. NIS-elements software was used to quantify IHC staining.

Table 2 –

Immunohistochemistry primary antibodies

1° Antibody	Animal	Dilution	Company	Catalog #
CLDN3	Rabbit	1:40	abCam	ab15102
CLDN7	Rabbit	1:100	Invitrogen	34–9100
CLDN15	Rabbit	1:20	Invitrogen	38–9200
OCLN	Rabbit	1:100	Invitrogen	71–1500
ZO-1	Rabbit	1:100	abCam	ab216880
VIM	Mouse	1:100	Sigma	V2258–100uL
ACTA2	Rabbit	1:100	abCam	ab5694
KIT	Rabbit	1:50	DAKO	A4502
CDH1	Mouse	1:200	BD Transduction Laboratories	610181

Table 3 –

Immunohistochemistry secondary antibodies

2° Antibody	Animal	Dilution	Company	Catalog #
Alexa Flour 488	Anti-rabbit	1:500	Invitrogen	A21206
Alexa Flour 568	Anti-rabbit	1:500	Invitrogen	A11011
Alexa Flour 647	Anti-rabbit	1:500	Invitrogen	A31573
Alexa Flour 488	Anti-mouse	1:500	Invitrogen	A21202
Alexa Flour 647	Anti-mouse	1:200	Invitrogen	A32787

Ussing chamber

8–11 week old tHIOs were harvested for ex vivo analysis in an Ussing Chamber (Warner Instruments). Recordings were made using an EC-825 Epithelial voltage clamp (Warner Instruments) connected to an iWorx 118 recording unit with Labscribe 4 (iWorx). Excised tHIOs were dissected and planarized in ice cold Kreb’s buffer solution, mounted between the hemi-chambers of an Ussing Chamber (pore surface area 0.11cm²), and maintained in 37◦C Kreb’s buffer, gassed with 95% O₂ + 5% CO₂. Transepithelial electrical resistance (TER) was measured by voltage clamping the membrane at 1mV and recording the current. Resistance was calculated using Ohms Law. Resistance in the system was accounted for by measuring the resistance in an empty chamber (R₀) and subtracting R₀ from the resistance of the samples. Short circuit current (Isc) was measured by voltage clamping the membrane to 0mV and recording the current. Epithelial permeability was measured by recording fluorescein isothiocyanate (FITC)-dextran flux through the tHIO. FITC-dextran permeability was measured by adding 2.2mg/mL of 4kDa FITC-dextran to the apical (luminal) side of the tHIO and taking a fluid sample from the basolateral side every 15 minutes for 1 hour. Samples were read with a microplate reader (Cytation 5, BioTek).

Contractility apparatus

12 week old tHIOs were harvested for ex vivo contractility studies in an organ bath. The tHIOs were cut into approximately 2mm × 4–9mm strips and these strips were mounted in 40 mL organ baths filled with Krebs buffer at 37°C and gassed with 95% O₂ + 5% CO₂. The isometric force was monitored by an external force displacement transducer connected to a biological recording system equipped with an amplifier (AD Instruments). Each strip was stretched to 0.5 gram tension and allowed to equilibrate for 30 minutes. After equilibration, basal contractile activity data was recorded for a period of 10 minutes for each tHIO strip. Once data acquisition was completed, each intestinal strip was measured and the tissue removed, dried, and weighed. Contractile activity parameters were calculated over 5 min of recorded data using LabChart software. The smoothing function was applied to the data at 100ms. Total contractile activity (TCA) was calculated as the area under the curve. Amplitude was calculated as the average cycle height. Total contractile activity and amplitude were normalized to tissue cross sectional area. Frequency was calculated as the average contractions per minute. Tension was calculated as the average force applied normalized to tissue weight. We utilized two samples of human intestine (female jejunum and male ileum) from our IRB-approved Pediatric Gastrointestinal Biobank (protocol #HSC-MS-20–0731) as positive controls for these experiments.

Quantification of murine host sex hormone levels

Blood was collected from 12 week old male (n=4) and female (n=4) NSG mice by cardiac puncture. Samples were allowed to clot at room temperature for 90 minutes before centrifugation at 2000 × g for 15 minutes at room temperature to separate the serum. The serum was stored at – 20⁰ C until use. Estradiol was measured by ELISA (American Laboratory Products Co) following the manufacturer’s instructions. Progesterone and testosterone were measured by ELISA (Immuno-Biological Laboratories, Inc) following the manufacturer’s instructions.

Statistical analysis

Continuous data are represented as mean ± standard error of the mean (SEM). Student’s tests were used to analyze continuous data. Statistical analyses were performed using Prism 9 (GraphPad Software). Level of significance was set to p<0.05.

Results

Sex-hormone levels were distinct in male versus female NSG mice.

In order to evaluate if circulating sex-hormones may be different in the 9–18 week old male and female NSG mice that are utilized as hosts for tHIOs, ELISA was used to measure serum progesterone, estradiol, and testosterone levels in 12 week old male (n=4) and female (n=4) NSG mice. We found that serum progesterone levels were significantly lower in males versus females (8.28±1.8056 vs all four females measuring >40 ng/dL over the reportable range of the ELISA kit), estradiol levels were not significantly different (77.85 vs 82.95 ng/dL, p=0.860, Supplemental Figure 1A), whereas serum testosterone levels were higher in males compared to females (882 vs 55.8 ng/dL, p=0.0214, Supplemental Figure 1B). This significantly different serum level of sex-hormones progesterone and testosterone in male versus female NSG mice suggests that there is a potential for sex-related differences when these NSG mice are utilized as hosts for the transplantation of HIOs.

The tHIO epithelial barrier formed by tight junctions is not influenced by murine host sex

In order to determine if there were sex-related differences in the establishment of the epithelial barrier in tHIOs, RT-qPCR and IHC was used to investigate the gene expression and morphology, respectively, of tight junctions in tHIOs grown in male and female murine hosts. RT-qPCR was used to investigate claudin 3 (CLDN3), claudin 7 (CLDN7), claudin 15 (CLDN15), occludin (OCLN), and tight junction protein-1, also known as zonula occludens-1 (TJP1). The tight junction is complex with over forty components (18, 21–23); thus, we chose to focus on those components which would significantly effect small intestinal epithelial barrier function (24) as previously described (4). We found that the expression of these select tight junction genes relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in tHIOs from male (n=10–12) and female (n=13–15) murine hosts was not significantly different (CLDN3, 0.87 vs 0.97, p=0.64; CLDN7, 0.99 vs 1.18, p=0.58; CLDN15, 0.95 vs 0.82, p=0.42; OCLN, 1.38 vs 1.11, p= 0.14; TJP1, 1.10 vs 1.05, p=0.90; Figure 1A–E). IHC for the analogous proteins revealed the expected paracellular distribution for all three claudins and characteristic apical staining for OCLN and ZO-1 in both groups with no differences in morphology (Figure 1F–J). Furthermore, we demonstrated that the expression of these select tight junction proteins relative to epithelial marker, E-cadherin (CDH1) in tHIOs from male (n=3–4) and female (n=3–5) murine hosts was not significantly different (CLDN3 1.09 vs 1.06, p=0.86, CLDN7 0.90 vs 1.09, p=0.47, CLDN15 0.94 vs 1.19, p=0.33, OCLN 0.43 vs 0.55, p=0.39, ZO-1 0.50 vs 0.31, p=0.16), consistent with the RT-qPCR results. This suggests that tight junction gene expression as well as protein morphology and expression in tHIOs grown in vivo is not altered by murine host sex.

Figure 1: Transplanted human intestinal organoids (tHIOs) from male and female hosts have comparable expression and localization of tight junction markers.

RT-qPCR demonstrates no difference in the expression of select tight junction genes claudin 3 (CLDN3), claudin 7 (CLDN7), claudin 15 (CLDN15), occludin (OCLN), and tight junction protein-1 (TJP1) relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) between tHIOs from male (n=10–12) and female (n=13–15) hosts (A-E). IHC staining shows no difference in the morphology and expression of the analogous tight junction proteins relative to ecadherin (CDH1) in tHIOs from male (n=3–4) and female (n=3–5) hosts (F-J). Error bars represent mean ± SEM, *p<0.05. Scale bars represent 50μm for F-J.

tHIO epithelial barrier function is not affected by murine host sex

To evaluate SABV in tHIO epithelial barrier function, an Ussing chamber was used to measure TER, Isc, and permeability in tHIOs grown in male and female murine hosts. TER (29.8 vs 23.5 Ω*cm², p=0.40) (Figure 2A) and Isc (7.698 vs 13.7 μA/cm², p=0.53) (Figure 2B) in male (n=4) and female (n=5) hosts, respectively, was not significantly different. FITC-dextran permeability over 60 minutes and quantified by the area under the curve (AUC) was, likewise, no different (365,060 vs 257,058 ng/mL/cm²/min, p=0.66) in male (n=4) and female (n=5) hosts (Figure 2C&D). This data suggests that tHIO epithelial barrier function is not influenced by murine host sex.

Figure 2: Epithelial barrier function is not altered in transplanted human intestinal organoids (tHIOs) from male and female hosts.

Transepithelial electrical resistance and short circuit current are not significantly different in tHIOs from male (n=4) vs female (n=5) hosts (A&B). A FITC-dextran permeability assay revealed no difference in tHIO epithelial permeability between tHIOs from male (n=4) vs female (n=5) hosts (C&D). Error bars represent mean ± SEM, *p<0.05.

Murine host sex does not alter mesenchymal components responsible for muscle contractility in tHIOs

To ascertain if murine host sex affects certain mesenchymal components responsible for muscle contractility of tHIOs, RT-qPCR was conducted for mesenchymal marker, VIM (vimentin), smooth muscle marker, ACTA2 (smooth muscle actin), and interstitial cells of Cajal (ICC), also known as pacemaker cells, which express KIT (a receptor tyrosine kinase) and IHC was performed for the corresponding proteins in tHIOs grown in male and female murine hosts. RT-qPCR revealed that the expression of VIM, ACTA2 and KIT relative to GAPDH in tHIOs from male (n=5–10) versus female (n=6–13) murine hosts was not significantly different (VIM 0.24 vs 0.30, p=0.61; ACTA2 0.30 vs 0.28, p=0.51; KIT 0.73 vs 0.92, p=0.34; Figure 3A–C). IHC revealed similar protein expression in tHIOs grown in vivo in male (n=3) and female (n=3) murine hosts (Figure 3D). This suggests that select mesenchymal components responsible for muscle contractility in tHIOs are not affected by murine host sex.

Figure 3: Transplanted human intestinal organoids (tHIOs) from male and female murine hosts have similar expression and localization of mesenchymal components responsible for muscle contractility.

RT-qPCR shows no difference in the expression of mesenchymal marker, VIM (vimentin), smooth muscle marker, ACTA2 (smooth muscle actin), and interstitial cells of Cajal (ICC), also known as pacemaker cells, which express KIT (a receptor tyrosine kinase) in male (n=5–10) vs female (n=6–13) hosts (A-C). IHC staining reveals no difference in morphology of the corresponding proteins in tHIOs from male (n=3) and female (n=3) hosts (D). CDH1 expression demonstrates the tHIO epithelium for reference (D). Error bars represent mean ± SEM, *p<0.05. Scale bars represent 100μm for D.

tHIO muscle contractility does not demonstrate host sex-related differences

In order to examine if muscle contractility in tHIOs exhibited sex-related differences, experiments measuring contractility of tHIOs from male and female murine hosts in an organ bath were conducted. tHIOs from male (n=7) and female (n=5) hosts displayed no difference in contractility (Figure 4A) as measured by TCA (1244 vs 529.8 g*s/cm², p=0.23), amplitude (1.76 vs 0.78 g/cm², p=0.17), frequency (11.0 vs 11.4 contractions/min, p=0.86) and tension (35.0 vs 48.6 g/g tissue, p=0.38), respectively (Figure 4B–E). This suggests that muscle contractility is not influenced by the host sex in which tHIOs are grown in vivo. As a positive control, muscle contractility was also evaluated in human intestine from male ileum (n=1) and female jejunum (n=1) as measured by TCA (466.9 vs 979.9 g*s/cm²), amplitude (0.22 vs 1.94 g/cm²), frequency (8.9 vs 9.7 contractions/min), and tension (3.7 vs 7.8 g/g tissue), respectively.

Figure 4: Muscle contractility is not significantly different in transplanted human intestinal organoids (tHIOs) from male and female murine hosts.

Representative contractile waveforms of tHIOs from male (red waveform) and female (blue waveform) hosts recorded over a 5-minute data analysis period (A). Total contractile activity of tHIOs from male vs female hosts is not significantly different (B). tHIOs from male vs female murine hosts exhibited comparable amplitude, contractile frequency, and tension (C-E). For all contractile data: male hosts n=7 and female hosts n=5. Error bars represent mean ± SEM, *p≤0.05.

Discussion

We aimed to investigate murine host SABV in tHIO epithelial barrier function and muscle contractility. We hypothesized that tHIOs from female hosts would exhibit superior intestinal epithelial barrier function, whereas tHIOs from male hosts would display enhanced muscle contractility. In this study, we demonstrated lower progesterone, equivalent estradiol, and higher testosterone serum levels in male versus female adult NSG mice (Supplemental Figure 1). Despite these distinct sex-hormone levels between male and female NSG mice, murine host sex does not influence the formation of the epithelial barrier in tHIOs. Select tight junction gene expression and protein morphology was no different in tHIOs in male and female murine hosts (Figure 1A–J). Furthermore, functional assays of the epithelial barrier demonstrated no significant difference in TER, Isc, nor FITC-dextran flux (Figure 2 A–D). Alongside our findings of comparable epithelial barrier maintenance in tHIOs from both hosts, muscle contractility was also not influenced by host sex. The gene and protein expression of mesenchymal markers for components responsible for muscle contractility were no different in tHIOs from male and female murine hosts (Figure 3A–D). Finally, functional examination of muscle contractility demonstrated no difference in TCA, amplitude, frequency, and tension in tHIOs from male and female murine hosts (Figure 4A–E).

These findings have important implications moving forward to advance and optimize the tHIO model, in particular, the success of HIO transplantation surgery. The transplantation of HIOs beneath the kidney capsule is delicate and technically challenging. Care taken during transplantation is critical for both murine host health and successful engraftment rates (10). Almost all prior studies, including ours, utilize male mice as the host for tHIO in vivo growth (3, 4, 7–12). One likely factor contributing to this discrepancy is that male mice are larger and therefore have larger kidney size versus age matched females. Utilizing male mice with larger kidneys may make HIO transplantation surgery beneath the kidney capsule easier (25). Thus, the results of our study suggest that continued use of male mice as the preferred host, to maximize both host survival as well as tHIO engraftment rates, will not influence the results obtained regarding tHIO epithelial barrier and muscle contractile function. In addition to these benefits, recent studies from our lab have demonstrated that tHIOs from male hosts are more likely to produce tHIOs with significantly larger lumens and a trend toward fewer lumens versus tHIOs from female hosts (26). The ability to generate tHIOs with a single large lumen is critical for a number of downstream applications such as functional experiments using an Ussing chamber and contractility apparatus, as well as studies that require a surgical anastomosis between the tHIO and host murine intestine (11). Furthermore, these investigations also demonstrated that tHIOs from male hosts generated epithelium with higher grades of development (26). Improved epithelial maturation is imperative not only for the tHIO model to serve as an ideal investigative platform that recapitulates human intestine, but also to bring the field one step closer to generating an HIO-derived tissue-engineered intestine that can serve as a therapy for children suffering from IF. Thus, taking these recently published findings as well as the results of this current study together, we cautiously conclude that male hosts appear to be the preferred host for tHIO transplantation surgery, and begin to establish assurance that functional readouts utilizing an Ussing chamber or contractility apparatus should remain unaffected by male host sex.

Though the data in our study suggests that murine host sex may not influence tHIO epithelial barrier function and muscle contractility there are limitations to our study that warrant proper discussion. First, although we measured significant differences in progesterone and testosterone levels in 12 week old NSG male and female mice, we did not measure sex hormone level fluctuations in the murine hosts throughout the 8–12 week period during which tHIOs grew in vivo. Due to this limitation, we are unable to ascertain whether specific levels or certain types of circulating sex hormones contributed to the results found in this study. Future studies should consider monitoring murine host sex hormone fluctuations during tHIO growth in both male and female hosts to determine if specific hormones or certain levels of hormones contribute to epithelial barrier or muscle contractile function. Performing ovariectomy in female hosts or orchiectomy in male hosts to eliminate specific sex hormones could also prove useful (27, 28). Second, the hESC cell line used to generate the HIOs in this study was an H9 cell line which is derived from an XX, female donor, and donor sex can contribute to variations between cell lines (29). Our studies should be repeated utilizing HIOs generated from an H1 cell line which is from an XY, male donor, to account for potential sex-related differences in hESC lines. Third, our reported TER values in tHIOs are lower than previously published (12). It is important to note that there can be a large variation in TER values ranging from 50 to 8,000 Ω*cm² for in vitro versus ex vivo studies, distinct cell lines, intestine from different species, as well as different regions of the intestine (e.g. jejunum vs ileum vs colon) (30). The only other research group to measure TER in tHIOs reported values ranging from approximately 90–280 Ω*cm² (12). Here we report TER in tHIOs ranging from 14 to 43 Ω*cm². The lower TER values measured in our tHIOs are challenging to interpret. There are several important differences between the studies conducted by Poling et al and our lab: they applied a villus correction factor for differences in villus size and we do not, they perform seromuscular stripping and we do not, and they utilized different Ussing chamber equipment including a smaller pore size. Importantly, we do not perform seromuscular stripping as we aim to preserve intramural neuromuscular activity to measure its effect on barrier function in future experiments. These notable differences may account for the lower TER values in tHIOs measured in our studies. Another possible explanation for the lower TER in our tHIOs is immaturity of the epithelium or perturbation of the epithelial monolayer. Fourth, our FITC-dextran permeability results are limited as the flux was measured over a shorter period of 60 minutes whereas Poling et al measured over 3 hours. The time period for paracellular flux studies utilizing FITC-dextran, Lucifer yellow, or ⁵¹Cr-EDTA in the intestine can vary, ranging from 60 min (31), 90 min (32), 2 hours (19) to 3 hours (12). Typically, longer time periods for flux measurement are more accurate, although some researchers report that this accuracy may not be sustained for thicker human tissue, especially when full-thickness preparations are utilized as we have here (33). Nonetheless, the interpretation of our FITC-dextran permeability data is potentially limited by this shorter flux period. Fifth, due to the limited availability of human intestinal samples from male and female donors as controls in our study, we are unable to ascertain whether epithelial barrier function or muscle contractility in tHIOs from male and female hosts shows pertinent differences compared to native human intestinal function as could be observed with a sufficiently powered human intestinal sample analysis. Future studies should aim to incorporate a higher number of male and female human intestinal samples as controls to ascertain these potential differences. Finally, the tHIOs used in this study did not have an enteric nervous system. It is known that the enteric nervous system (ENS), enteric glial cells specifically, play a significant role in both maintaining the epithelial barrier and coordinating muscle contractility in the intestine. Future studies should consider how incorporation of an ENS into the tHIO model could influence potential murine host sex-related differences on both the epithelial barrier and muscle contractility.

In conclusion, our data suggests the murine host sex may not be a significant biological variable in the epithelial barrier and muscle contractile function of tHIOs. Though future studies will need to further isolate the many variables that contribute to the host sex influence on tHIO development and function, and also address the limitations identified in this study, the tHIO model remains a promising developing technology in the generation of human intestine that may be a potential cure for the many suffering from IF across the world.

Nonstandard Abbreviations:

HIOs

human intestinal organoids

tHIOs

transplanted human intestinal organoids

SABV

sex as a biological variable

IF

intestinal failure

PN

parenteral nutrition

IBD

inflammatory bowel disease

NSG

nonobese diabetic severe combined immunodeficiency gamma chain deficient

hESCs

human embryonic stem cells

PFA

paraformaldehyde

RT

room temperature

H&E

hematoxylin and eosin

TER

transepithelial electrical resistance

ISC

short circuit current

FITC

fluorescein isothiocyanate

TCA

total contractile activity

SEM

standard error of the mean

AUC

area under the curve

ICC

interstitial cells of Cajal

ENS

enteric nervous system

Data Availability Statement:

The data that support the findings of this study are available in the methods and/or supplementary material of this article. Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.