A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells

Abstract

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung, liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells, hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2, SOX17, HNF1B, members of the GATA family, and the surface receptor CXCR4. However, differentiation protocols are rarely 100% efficient. Here, we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations.

Anterior–Posterior Patterning of Definitive Endoderm Generated from Human Embryonic Stem Cells Depends on the Differential Signaling of Retinoic Acid, Wnt-, and BMP-Signaling

“…The MACS enrichment resulted in a substantially more viable and greater cell population. Thus, we opted for the MACS technique for all further experiments since this technique resulted in a proper purity and good viability after reseeding for further experimentation .…”

“…MACS was performed on day 3 or 4 of the endoderm differentiation protocol. The success of the sorting was monitored by flow cytometric analysis of the CXCR4-staining before and after the procedure [21]. Purified CXCR4-positive cells were then reseeded on 12-well or 6-well plates in a density of 110,000-170,000 cells per cm 2 in advanced RPMI 1640 with 50 ng/ml activin A, 1% penicillin/ streptomycin, 1% glutamax, 0.2% FBS, and 5-10 mM Rock inhibitor for 24 hours before changing to the inductive media conditions as outlined above.…”

“…The MACS enrichment resulted in a substantially more viable and greater cell population. Thus, we opted for the MACS technique for all further experiments since this technique resulted in a proper purity and good viability after reseeding for further experimentation [20,21]. DE-cells were MACS-sorted via the surface antigen CXCR4 to exclude CXCR4-negative cells from further differentiation, which did not adequately respond to the directed differentiation procedure [20,21].…”

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).…”