Long-term renewable human intestinal epithelial stem cells as monolayers: A potential for clinical use

Andrew Scott; Joshua D Rouch; Ziyad Jabaji; Hassan A Khalil; Sergio Solorzano; Michael Lewis; Martín G Martín; Matthias G Stelzner; James C Y Dunn

doi:10.1016/j.jpedsurg.2016.02.074

Long-term renewable human intestinal epithelial stem cells as monolayers: A potential for clinical use

J Pediatr Surg. 2016 Jun;51(6):995-1000. doi: 10.1016/j.jpedsurg.2016.02.074. Epub 2016 Mar 2.

Authors

Andrew Scott¹, Joshua D Rouch¹, Ziyad Jabaji¹, Hassan A Khalil¹, Sergio Solorzano², Michael Lewis³, Martín G Martín⁴, Matthias G Stelzner⁵, James C Y Dunn⁶

Affiliations

¹ Department of Surgery, Division of Pediatric Surgery, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA.
² 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.
³ Veterans Affairs Greater Los Angeles Healthcare System, Department of Pathology, Los Angeles, CA.
⁴ 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.
⁵ Veterans Affairs Greater Los Angeles Healthcare System, Department of Surgery, Los Angeles, CA.
⁶ 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.

Abstract

Purpose: 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.

Conclusion: This 2D system is an important step to expand hISCs for further experimental studies and for clinical cell transplantation.

Level of evidence: 1 Experimental.

Keywords: Differentiation; Enteroids; Human intestinal epithelial stem cells; Human laminin; In vitro culture; Monolayers; Spheroids; Type I collagen.

MeSH terms

Biocompatible Materials
Cadaver
Cell Culture Techniques
Collagen Type I
Epithelial Cells / cytology*
Humans
In Vitro Techniques
Intestinal Mucosa / cytology*
Laminin
Stem Cells / cytology*

Substances

Biocompatible Materials
Collagen Type I
Laminin

Abstract

MeSH terms

Substances

Grants and funding