Cd133

STCs were isolated from fresh pig kidneys, as previously shown [7 (link),12 (link)]. Briefly, pig cortical and medullary sections were washed with phosphate-buffered saline, diced, and digested with 2 mg/mL collagenase for 1 h. Samples were then forced through a 60-mesh (250 μm) steel sieve to remove the fibrous component [34 (link)]. The cellular fraction was passed through 100 μm cell strainer followed by the addition of Medium 199 containing 3% FBS (Gibco BRL, Waltham, MA, USA) [35 (link)] at 37 °C in a humidified atmosphere with 5% CO2. The culture medium was replaced every 2 days to remove non-adherent cells. Two weeks later, the adherent cells were harvested with TrypLE™ Express (Gibco) treatment and sub-cultured. Cultured pig STCs were characterized using flow cytometry and immunofluorescence staining, which confirmed their positivity for CD133 (Novus Biologicals, Centennial, CO, USA) and CD24 (Abcam, San Francisco, CA, USA).

Human skin tumor tissue microarrays were purchased from US Biomax (SK801b; Rockville, MD, USA). Paraffin-embedded human and mouse tumor tissue sections were blocked for 60 min with 2% normal horse or goat serum diluted in PBS. The sections were then blotted and incubated with specific primary antibodies in blocking serum for overnight at 4 °C. The next day, the slides were washed three times for 5 min each in PBS and incubated in biotinylated anti-mouse, rabbit, rat or goat antibody for 1 h. The slides were washed in PBS, followed by formation of the avidin-biotin-peroxidase complex (ABC, Vector Laboratories, Inc., Burlingame, CA, USA). The slides were washed, and the peroxidase reaction developed with diaminobenzidine and peroxide, then counterstained with hematoxylin, mounted in Cytoseal XYL (Thermo Fisher Scientific, Waltham, MA, USA), and evaluated using a light microscope (× 200, Olympus, Tokyo, Japan). Specific primary antibodies were purchased from Santa Cruz Bio (PCNA, F4/80 and p50; Dallas, TX, USA), eBioscience (Ly6G and CD11b; Thermo Fisher Scientific, Waltham, MA, USA), Abnova (CD133; Taipei, Taiwan), Novus Biologicals (TIMP-1; Littleton, CO, USA) and Abcam (IL-32, CD44, ITGAV and p65; Cambridge, MA, USA).

For in vitro studies, pig proximal kidney TEC (LLC-PK1, ATCC, Manassas) were cultured in Medium-199 (Gibco BRL, USA) containing 3% FBS^{15 (link)}. STC-like cells were isolated from fresh pig kidneys (6 month old, normal diet) as previously described^{34 (link)}, with few modifications. Briefly, 3–5 g pig kidneys including both cortex and medulla were sectioned and washed with PBS. Kidneys were diced and digested with 2 mg/ml collagenase for 1 hour, then forced through a 60-mesh (250-μm) steel sieve to remove the fibrous component. The cellular fraction was then passed through 100 μm cell strainer and followed by the addition of Medium 199 containing 3% fetal bovine serum (Gibco BRL, USA) at 37 °C in a humidified atmosphere with 5% CO₂. The culture medium was replaced every 2 days to remove non-adherent cells. After about two weeks, the adherent cells were harvested with 0.25% trypsin (Gibco BRL, USA) treatment and sub-cultured. Phenotypic analysis of cultured STC-like cells (as well as PK1 TEC) employed immunofluorescence for CD133 (Novus Biologicals), CD24 (Abcam), KIM1 (R&D Systems), Vimentin (Abcam), and OCT4 (Abcam).

All protocols were approved by the Mayo Clinic Institutional Animal Care and Use Committee (protocol # A1609-16), all experiments were performed in accordance with relevant guidelines and regulations. STC-like cells acquired from domestic pig kidneys were used for the studies. Frozen kidney sections from 2 pigs (6 month old, normal diet) were used for double-staining of CD133 (1:100, Novus Biologicals) and CD24 (1:100, Abcam) to detect resident STC-like cells, and phaseolus vulgaris erythroagglutinin (PHA-E, Invitrogen) as a proximal tubular marker.
Male 129-S1 mice (Jackson Lab, ME, 11 weeks of age) were studied for 4 weeks, randomly divided into Sham (n = 8), RAS + Vehicle (n = 10), and RAS + STC-like cells-EV (n = 10) groups. RAS was induced by surgical placement of a 0.15 mm diameter arterial cuff, whereas sham surgeries without placement of a cuff were performed in the control group, as previously described³³ . After 2 weeks the carotid artery was cannulated via a vascular cut down, and 200 uL PBS or STC-like cells-EV (30 ug in 200 uL PBS) slowly injected caudally. In some mice, the STK was obtained from Mito-Tracker or CM-Dil labeled EV-treated mice for EV tracking 2 weeks after injection (n = 4 each group). All remaining mice were scanned with MRI, 2 weeks after injection, and subsequently euthanized with CO₂. Kidneys and blood samples were collected for ex-vivo studies.

STC activation was assessed by counting the total number of STCs in pig kidney sections using flow cytometry (FlowSight, Amnis, Seattle, WA, USA) with CD133 (1:25, Novus Biologics, Centennial, CO, USA cat#: NB120-16518G) CD24 (1:25, Abcam, cat#: ab134375) antibodies [12 (link)]. At least 100,000 events were acquired. Following particle focusing, doublets, cell clusters, and debris were eliminated by gating on events with a high aspect ratio (>0.6) and for cell size (10–20 µm diameter, 100 to 400 µm² area) using bright field area vs. aspect ratio features. CD133 and CD24 positive events were quantified using flow-gaiting strategy: number of double-positive CD133 and CD24 events/number of singlet events between 10–20 µm diameter and expressed as a percentage. To detect STCs in frozen pig kidney sections, we performed double-staining of CD133 (1:100, Novus Biologicals, Centennial, CO, USA) and CD24 (1:100, Abcam, San Francisco, CA, USA), and the tubular marker phaseolus vulgaris erythroagglutinin (PHA-E, Invitrogen, Carlsbad, CA, USA) [12 (link)].

Immunofluorescence staining were done as previously described^{33 (link)}. CD133 was purchased from Novus Biologicals (Littleton, CO, USA). pSTAT5 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Skin tissues from WT mice and IL-32γ mice were lysed by Pro-prep protein extraction buffer (iNtRON, Sungnam, Korea) and the total protein concentration was determined using the Bradford reagent (Bio-Rad, Hercules, CA, USA). Nuclear extraction was performed using nuclear extraction kit (Abcam, Cambridge, MA, USA). The membranes were immunoblotted with specific primary antibodies. The intensity of the bands was measured using the Fusion FX 7 image acquisition system (Vilber Lourmat, Eberhardzell, Germany). Specific primary antibodies were purchased from Santa Cruz Bio (p-IKKα/β, IKKα/β, p-JNK, JNK, p-ERK, p-p38, p38, p-STAT3, STAT3, p50, Histone H1, Cyclin D1, CDK4, Bax, Bcl-2, MMP-2, MMP-9 and β-actin; Dallas, TX, USA), Cell Signaling Technology (Myc-tag and ERK; Trask Lane, Danvers, MA, USA), Abnova (CD133; Taipei, Taiwan), Novus Biologicals (TIMP-1, iNOS and COX-2; Littleton, CO, USA) and Abcam (CD44, ITGAV, S100A8 and p65; Cambridge, MA, USA). β-actin and Histone H1 was used as a loading control.