title: Long-term renewable human intestinal epithelial stem cells as monolayers: A potential for clinical use.
abstract: OBJECTIVE
  Current culture schema for human intestinal stem cells (hISCs) frequently rely on a 3D culture system using Matrigel™, a laminin-rich matrix derived from murine sarcoma that is not suitable for clinical use. We have developed a novel 2D culture system for the in vitro expansion of hISCs as an intestinal epithelial monolayer without the use of Matrigel.
  
  
  METHODS
  Cadaveric duodenal samples were processed to isolate intestinal crypts from the mucosa. Crypts were cultured on a thin coat of type I collagen or laminin. Intestinal epithelial monolayers were supported with growth factors to promote self-renewal or differentiation of the hISCs. Proliferating monolayers were sub-cultured every 4-5days.
  
  
  RESULTS
  Intestinal epithelial monolayers were capable of long-term cell renewal. Less differentiated monolayers expressed high levels of gene marker LGR5, while more differentiated monolayers had higher expressions of CDX2, MUC2, LYZ, DEF5, and CHGA. Furthermore, monolayers were capable of passaging into a 3D culture system to generate spheroids and enteroids.
  
  
  CONCLUSIONS
  This 2D system is an important step to expand hISCs for further experimental studies and for clinical cell transplantation.
  
  
  METHODS
  1 Experimental.
authors.affiliation: Department of Surgery, Division of Pediatric Surgery, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Scott
authors.affiliation: Department of Surgery, Division of Pediatric Surgery, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA.
authors.forename: Joshua D
authors.initials: JD
authors.lastname: Rouch
authors.affiliation: Department of Surgery, Division of Pediatric Surgery, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA.
authors.forename: Ziyad
authors.initials: Z
authors.lastname: Jabaji
authors.affiliation: Department of Surgery, Division of Pediatric Surgery, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA.
authors.forename: Hassan A
authors.initials: HA
authors.lastname: Khalil
authors.affiliation: Department of Pediatrics, Division of Gastroenterology and Nutrition, Mattel Children's Hospital and the David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA.
authors.forename: Sergio
authors.initials: S
authors.lastname: Solorzano
authors.affiliation: Veterans Affairs Greater Los Angeles Healthcare System, Department of Pathology, Los Angeles, CA.
authors.forename: Michael
authors.initials: M
authors.lastname: Lewis
authors.affiliation: Department of Pediatrics, Division of Gastroenterology and Nutrition, Mattel Children's Hospital and the David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA.
authors.forename: Martín G
authors.initials: MG
authors.lastname: Martín
authors.affiliation: Veterans Affairs Greater Los Angeles Healthcare System, Department of Surgery, Los Angeles, CA.
authors.forename: Matthias G
authors.initials: MG
authors.lastname: Stelzner
authors.affiliation: Department of Surgery, Division of Pediatric Surgery, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA; School of Engineering, Department of Bioengineering, University of California, Los Angeles, Los Angeles, CA. Electronic address: jdunn@mednet.ucla.edu.
authors.forename: James C Y
authors.initials: JC
authors.lastname: Dunn
mesh_terms: D001672:Biocompatible Materials; D002102:Cadaver; D018929:Cell Culture Techniques; D024042:Collagen Type I; D004847:Epithelial Cells / Q000166:cytology*; D006801:Humans; D066298:In Vitro Techniques; D007413:Intestinal Mucosa / Q000166:cytology*; D007797:Laminin; D013234:Stem Cells / Q000166:cytology*
chemical_list: D001672:Biocompatible Materials; D024042:Collagen Type I; D007797:Laminin
keywords: Differentiation; Enteroids; Human intestinal epithelial stem cells; Human laminin; In vitro culture; Monolayers; Spheroids; Type I collagen