Stem Cell Biology and Research

Stem Cell Biology and Research

ISSN 2054-717X
Original Research

Directed differentiation protocols for successful human intestinal organoids derived from multiple induced pluripotent stem cell lines

Amanda L. Kauffman1, Jason E. Ekert1, Alexandra V. Gyurdieva1, Michael A. Rycyzyn1 and Pamela J. Hornby2*

*Correspondence: Pamela J. Hornby

2. Cardiovascular Metabolic Research, Janssen Pharmaceutical J&J, Spring House, PA 19477, USA.

Author Affiliations

1. Biologics Research, Biotechnology CoE, Janssen Pharmaceutical J&J, Spring House, PA 19477, USA.


Background: Relatively little has been reported comparing the ability of different induced pluripotent stem cells (iPSCs) and protocols to derive human intestinal organoids (HIO), although there is potential to supply HIO for translational research and regenerative medicine. In view of the time and effort required to differentiate HIO, protocols for differentiation were compared and five iPSC lines, produced by retroviral or non-viral reprogramming methods, were concurrently differentiated to HIO to evaluate the robustness and repeatability of using cellular markers by flow cytometry and immunohistochemistry.

Methods: iPSCs were differentiated to definitive endoderm by Activin A treatment (Protocol I) or Myostatin (GDF8), GSK-3β inhibitor (CHIR99021) and B27 supplement (Protocol II) and then differentiated to hindgut using FGF4 and WNT3A (Protocol I), or KGF and Retinoic Acid (Protocol II) resulting in spheroids that were differentiated to HIO in 3-dimensional culture in matrigel containing EGF, Noggin, and R-Spondin1.

Results: Definitive endoderm markers (CXCR4 and SOX17) were similar using both protocols and in all cell lines, but in their absence, spheroids to not develop. HIO derived from excised hindgut spheroids (Protocol II) showed more consistent expression of epithelial markers E-Cadherin, CDX2, villin, and chromogranin A. Morphological characteristics such as budding are predictive of robust enterocyte differentiation (villin positive) in HIO.

Conclusions: Substituting different members of the growth factor families (e.g., TGFβ) is equally or more effective for producing epithelial lineages in HIO. Although there are no early quantitative predictors of level of success, all reprogrammed somatic cells could be differentiated to produce HIO. Several morphological characteristics (excisable spheroids and budding in HIO) were associated with HIO success. This could improve cost-effectiveness and ease of differentiation as HIO become more commonly available as translational 3D models of the gut.

Keywords: Induced pluripotent stem cell, intestine, spheroid, human intestinal organoid

ISSN 2054-717X
Volume 2
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