Systematically labeling developmental stage-specific genes for the study of pancreatic β-cell differentiation from human embryonic stem cells

Abstract

The applications of human pluripotent stem cell (hPSC)-derived cells in regenerative medicine has encountered a long-standing challenge: how can we efficiently obtain mature cell types from hPSCs? Attempts to address this problem are hindered by the complexity of controlling cell fate commitment and the lack of sufficient developmental knowledge for guiding hPSC differentiation. Here, we developed a systematic strategy to study hPSC differentiation by labeling sequential developmental genes to encompass the major developmental stages, using the directed differentiation of pancreatic β cells from hPSCs as a model. We therefore generated a large panel of pancreas-specific mono- and dual-reporter cell lines. With this unique platform, we visualized the kinetics of the entire differentiation process in real time for the first time by monitoring the expression dynamics of the reporter genes, identified desired cell populations at each differentiation stage and demonstrated the ability to isolate these cell populations for further characterization. We further revealed the expression profiles of isolated NGN3-eGFP(+) cells by RNA sequencing and identified sushi domain-containing 2 (SUSD2) as a novel surface protein that enriches for pancreatic endocrine progenitors and early endocrine cells both in human embryonic stem cells (hESC)-derived pancreatic cells and in the developing human pancreas. Moreover, we captured a series of cell fate transition events in real time, identified multiple cell subpopulations and unveiled their distinct gene expression profiles, among heterogeneous progenitors for the first time using our dual reporter hESC lines. The exploration of this platform and our new findings will pave the way to obtain mature β cells in vitro.

Using stem cells to produce insulin

“…To address these limitations, homologous recombination to 'knock in' a fluorescent protein into a specific genetic locus can be used. Currently, hESC reporter lines exist that have been used to sort neuronal [145], cardiac [146] and b-cell precursors [147,148].…”

Bringing Stem Cell‐Based Therapies for Type 1 Diabetes to the Clinic: Early Insights from Bioprocess Economics and Cost‐Effectiveness Analysis

“…In our model, the current low DSP yield reported for MACS purification of islet cells limited the batch size as a theoretical maximum of just above 5 billion differentiated islet cells (i.e., enough doses for ten patients) can be obtained per MACS cycle . Increasing the MACS yield to values reported for affinity purification processes for pancreatic progenitors and definitive endoderm yielded significant cost‐savings per dose. Current clinical trials do not employ an affinity purification step and encapsulate the pancreatic progenitors assuming that a very high differentiation efficiency is sufficient to minimize the occurrence of teratomas.…”

“…We simulated a DSP strategy using magnetic‐activated cell sorting (MACS). This technique was previously reported for the purification of β cells from a complex mixture as well as for the positive selection of pancreatic and endoderm progenitors derived from stem cells . A DSP yield of 20% was used for model runs, based on the yield from the purification of β cells from cadaveric pancreatic donors (Figure S1 and Table S1, Supporting Information).…”

The quest to make fully functional human pancreatic beta cells from embryonic stem cells: climbing a mountain in the clouds

“…CRISPR and other genome-editing tools will soon permit mechanistic interrogation of non-human primates and other large animal models, as well as stem cells. In vitro studies will be augmented by inserting reporters into stem cell genomes [61] and by non-invasive imaging [62]. Moreover, in vitro culture systems can be designed to mimic the in vivo situation, including the intricate three-dimensional structure of the developing islet and/or pancreas.…”