# A retinoid analogue, TTNPB, promotes clonal expansion of human pluripotent stem cells by upregulating CLDN2 and HoxA1

## TL;DR

A retinoid called TTNPB helps human stem cells grow better in clonal cultures by boosting cell adhesion and renewal.

## Contribution

TTNPB synergizes with Y-27632 to dramatically improve hPSC cloning efficiency and expansion.

## Key findings

- TTNPB increases hPSC cloning efficiency from 0.2% to 20% when combined with Y-27632.
- TTNPB-treated cells show higher aggregate formation and proliferation in suspension culture.
- TTNPB promotes adhesion and self-renewal via upregulation of Claudin 2 and HoxA1.

## Abstract

Enzymatic dissociation of human pluripotent stem cells (hPSCs) into single cells during routine passage leads to massive cell death. Although the Rho-associated protein kinase inhibitor, Y-27632 can enhance hPSC survival and proliferation at high seeding density, dissociated single cells undergo apoptosis at clonal density. This presents a major hurdle when deriving genetically modified hPSC lines since transfection and genome editing efficiencies are not satisfactory. As a result, colonies tend to contain heterogeneous mixtures of both modified and unmodified cells, making it difficult to isolate the desired clone buried within the colony. In this study, we report improved clonal expansion of hPSCs using a retinoic acid analogue, TTNPB. When combined with Y-27632, TTNPB synergistically increased hPSC cloning efficiency by more than 2 orders of magnitude (0.2% to 20%), whereas TTNPB itself increased more than double cell number expansion compared to Y-27632. Furthermore, TTNPB-treated cells showed two times higher aggregate formation and cell proliferation compared to Y-27632 in suspension culture. TTNPB-treated cells displayed a normal karyotype, pluripotency and were able to stochastically differentiate into all three germ layers both in vitro and in vivo. TTNBP acts, in part, by promoting cellular adhesion and self-renewal through the upregulation of Claudin 2 and HoxA1. By promoting clonal expansion, TTNPB provides a new approach for isolating and expanding pure hPSCs for future cell therapy applications.

A retinoic acid analogue, TTNPB, improves clonal expansion in adherent and suspension culture of hPSCs by promoting cellular adhesion and self-renewal through the upregulation of Claudin 2 and HoxA1.