Isolation of esophageal stem cells with potential for therapy

Abstract

Purpose: Long-gap esophageal atresia represents a significant challenge for pediatric surgeons and current surgical approaches are associated with significant morbidity. A tissue-engineered esophagus, comprising cells seeded onto a scaffold, represents a therapeutic alternative. In this study, we aimed to determine the optimal techniques for isolation and culture of mouse esophageal epithelial cells and to isolate CD34-positive esophageal epithelial stem cells from cadaveric mouse specimens.

Methods: Primary epithelial cells were isolated from mouse esophagi by enzymatic dissociation from the mucosal layer (Dispase, Trypsin/EDTA) using three different protocols. In protocol A, isolated mucosa was minced and incubated with trypsin once. In protocol B, intact mucosal sheets underwent two trypsin incubations yielding a single-cell suspension. In protocol C, intact mucosa explants were plated epithelial side down. Epithelial cells were cultured on collagen-coated wells.

Results: Initial findings showed that Protocol B gave the best results in terms of yield, viability, and least contamination with different cell types and microbes. Esophageal epithelial cells isolated using Protocol B were stained for CD34 and sorted using fluorescence-activated cell sorting (FACS). Of the total cells sorted, 8.3% (2-11.3) [%median (range)] were CD34 positive.

Conclusions: Our results demonstrate that mouse esophageal epithelial cells can be successfully isolated from fresh mouse esophagi using two consecutive trypsin incubations of intact mucosal sheets. Furthermore, the cells obtained using this method were successfully stained for CD34, a putative esophageal epithelial stem cell marker. Further research into the factors necessary for the successful proliferation of CD34 positive stem cell lines is needed to progress toward clinical application.

Fig. 1

Esophageal TE requires the combination of appropriate scaffolds and cells. Cells used for repopulation of the epithelial and muscular layers can be derived from ESC, iPS, AFSC, and ASC; ESC embryonic stem cells, iPS induced pluripotent stem cells, AFSC amniotic fluid stem cells, ASC adult stem cells

Fig. 2

Collagen coating of plates (wet or dried) allowed a higher number of isolated cells to attach and grow following protocol A as shown by the light microscopy images (a). In the wells with no collagen spindle-shaped cells were mostly seen (inset). Pan-cytokeratin staining for epithelial cells demonstrated the epithelial identity of cells seeded onto collagen-coated plates, with no positive staining in the lack of collagen (b); scale bar 100 μm

Fig. 3

Cells isolated using protocol B showed a characteristic epithelial cobblestone morphology in culture (a), forming a monolayer by 2 weeks (b); scale bar 100 μm

Fig. 4

In protocol C, whole mucosal sheets were isolated (a), the mucosal side was identified by the mucosal ridges (b) and plated. Cells were seen migrating out of the mucosal sheet (c, asterisk) and acquiring cobblestone morphology (d). In 33 % of isolations, epithelial colonies were identified after 6 days (e), while 67 % of isolations were unsuccessful in doing so (f). In all cases, there was concomitant migration and attachment of spindle-shaped cells that did not stain positive for PCK-26 (g, h); scale bar 100 μm

Fig. 5

CD34 FACS sorting gates for CD34 dim (*) and bright (**) populations

Fig. 6

CD34⁺esophageal epithelial cells were sorted onto mitotically inactivated Swiss 3T3 fibroblasts. Dim and bright subpopulations showed similar growth characteristics in vitro, with Rhodamine B staining confirming colony formation at 2 weeks; scale bar 100 μm