Here, we describe a method for the purification of differentiated human embryonic stem cells that are committed towards the definitive endoderm for the improvement of downstream applications and further differentiations.
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.
Pluripotent stem cells such as embryonic stem cells (ESCs) have the capability to differentiate into virtually any cell type of the human body. Thus, in vitro differentiation protocols can be used to generate numerous adult cell types such as cardiomyocytes1, hepatocytes2, beta cells3, lung epithelial4 or neuronal cells5. This makes ESCs a valuable tool for the potential treatment of various degenerative diseases3.
The in vitro differentiation of ESCs towards adult tissues of the lung, liver and pancreas requires a pseudo-gastrulation into cells reminiscent of the definitive endoderm (DE)6. Since downstream differentiation towards the aforementioned somatic cell types is significantly less efficient, an optimal endoderm differentiation is regarded as rate-limiting7. Cells that are committed towards the endoderm lineage undergo characteristic changes in their gene expression profile. Pluripotency master regulator genes are down regulated, whereas the expression of other transcription factors such as FOXA2, SOX17, HNF1B, members of the GATA family and the surface receptor CXCR4 is highly upregulated6, 8, 9. CXCR4 is known to be transactivated by SMAD2/3, downstream of Nodal/TGF-β signaling and SOX17 due to specific binding sites in its promoter region10. Thus it is a very suitable marker used in a number of reports6, 8, 11-13. These expression changes reflects a pseudo-gastrulation event, in which ESCs first acquire characteristics of a primitive streak-like cell population and subsequently commit into the endoderm germ layer6.
However, differentiation protocols are rarely 100% efficient as a few cells may resist the differentiation process or differentiate towards other unintended lineages14. These cells may negatively influence further differentiation. Furthermore, residual undifferentiated cells harbor great risks for later transplantation experiments and may give rise to teratomas15-17.
To remove these unwanted cells early-on the surface marker CXCR4 can be used for the purification of cells that are committed towards the DE18. Here, we describe a method for the positive selection of CXCR4+ cells from DE differentiation cultures. For this, the surface marker CXCR4 is bound by an antibody which then in turn binds to magnetic microbeads. Unlike the harsh conditions during FACS sorting, the magnetically labeled DE-like cells can then easily be purified in a benchtop format using a gentle purification method. This protocol provides a straightforward method for the removal of cell populations that resisted the DE differentiation process.
1. Differentiation of Human ESC towards the Definitive Endoderm
2. Staining of CXCR4+ Definitive Endoderm Cells for Flow Cytometry Analysis
3. Magnetic Separation of CXCR4+ Cells
4. Optional: Analysis of Purified Definitive Endoderm Population
Upon differentiation ESCs undergo drastic changes in gene and protein expression. Figure 1 depicts typical marker genes that can be used to verify a successful endoderm differentiation. Prime targets for a gene expression analysis are GSC, FOXA2, and SOX17. In a relative gene expression analysis especially FOXA2 and SOX17 are increased by > 2,000 fold when compared to undifferentiated ESCs. GSC is already expressed very early within 24 hr during primitive streak formation but it is nonetheless induced > 100 fold on day four. The expression of these genes is consequently increased upon sorting using the CXCR4 surface marker6. Pluripotency master regulators such as OCT3/4 (POU5F1) and NANOG are significantly down regulated although the expression of these genes is still detectable after four days. This indicates that some cells do not adequately respond to the treatment regimen with CHIR-99021 and activin A. SOX7 gene expression is typically addressed to discriminate extra-embryonic endoderm formation against DE. In the first case SOX17 is co-expressed with SOX7 and in the latter case SOX17 is co-expressed with FOXA2. The results in Figure 1 indicate that along with DE some extra-embryonic cells may have formed during differentiation, although we have not yet been able to stain SOX7+ cells.
Figure 2 illustrates the different cell populations that are present after the differentiation of ESCs into the DE. In Step 1 ESCs are seeded as single cells and then differentiated by treatment with CHIR-99021 and activin A for four days. The IF staining of the DE markers SOX17 and FOXA2 (Figure 2A) shows that both markers are uniformly co-expressed within the nuclei. This is regarded as a hallmark of DE commitment. However, some cells resist the differentiation process and express neither of the two DE marker proteins (Figure 2A). In fact, two distinct cell populations are observed after the four day differentiation. While many cells express the DE marker FOXA2 there are still cells remaining that express the pluripotency marker SOX2 (Figure 2B). These two proteins are expressed in two separate populations and no co-expression can be observed (Figure 2B).
On day three or four of differentiation the surface protein CXCR4 is used to sort the DE-committed CXCR4+ population and thereby remove the remaining pluripotent cells and the other unwanted lineages. Depending on the differentiation efficiency of the used cell line > 80% CXCR4+ cells can be obtained using the differentiation protocol outlined in Step 18. The HES3 line used here yielded 66% ± 3% CXCR4-positive cells before sorting and 92% ± 4% after sorting (n=3). Figure 3 depicts flow cytometry data after staining and MACS purification of CXCR4+ cells after differentiation towards DE and shows the expression of common DE and pluripotency marker proteins using immunofluorescence (IF) staining before and after MACS purification. On day three of differentiation roughly 60% CXCR4+ cells using the immunofluorescence staining for CXCR4 described in Step 2 were obtained (Figure 3A, middle panel). CXCR4+ cells move from Q4 to Q1 upon staining with an APC conjugated anti-CXCR4 antibody. After the MACS purification described in Step 3 the CXCR4+ population is enriched to 85%. The purification process, however, does not eliminate all unwanted lineages from the cultures (Figure 3A, right panel).
After the MACS purification, the CXCR4+ cells can be seeded for further analysis or optionally a second round of sorting may be performed to yield higher purities but reduced viability. Prior to differentiation the wide-spread expression of the pluripotency marker SOX2 (stained in green) is detected by IF staining. In contrast, the DE marker FOXA2 (stained in red) cannot be detected (Figure 3B). In Figure 3C CXCR4+ cells are stained with antibodies for FOXA2 (green) and are co-stained for the pluripotency marker SOX2 (red). After seeding of the purified CXCR4+ cells only few SOX2+ cells are detected. The majority of the seeded cells are positive for the DE marker FOXA2.
Figure 1. Representative changes in gene expression of different pluripotency and endoderm marker genes during a four day endoderm differentiation. (A) Illustration of the protocols being used for endoderm differentiation followed by gene expression analysis. Random denotes to culture conditions without any growth factors, RP denotes to the differentiation using a reference protocol7, A a treatment with activin A alone, and CA-A (CHIR-99021 and activin A) the protocol, which is used for MACS sorting (see details in 8). The media used in the CA-A protocol are here referred as primitive streak induction medium and endoderm induction medium. (B) Depicted is the gene expression measured by RT-qPCR of GSC, SOX17 and FOXA2, which are expressed upon definitive endoderm commitment without further MACS sorting. POU5F1 (OCT3/4) and NANOG are pluripotency regulators and typically down regulated upon differentiation, whereas SOX7 is expressed in extra-embryonic endoderm. The gene expression was normalized against three stably expressed housekeeping genes (TBP, TUBA1A, G6PD) resulting in CNRQ values. The expression of the above mentioned genes in undifferentiated cells was set to 1 and changes are plotted as fold change of gene expression. Values are means ± SEM, n = 4-5. ANOVA plus Bonferroni's post hoc test. **P ≤ 0.01 compared with Random and #P ≤ 0.05, ##P ≤ 0.01 compared with RP (all data within this figure have recently been published in 8). Please click here to view a larger version of this figure.
Figure 2. Different cell populations after endoderm differentiation. (A) Staining of the DE markers SOX17 and FOXA2 with respective antibodies on d4 of differentiation reveals their homogenous co-localization. However, some cells resisted the differentiation process and do not express these markers (blue nucleus staining only). The merge of green and red staining is shown in yellow. (B) After DE differentiation there are two distinct cell populations. DE-like cells express FOXA2 whereas cells that resisted the differentiation process still express the pluripotency marker SOX2. (A-B) Nuclei were counterstained with DAPI (blue). Scale bar in panel (A) is 50 µm and in panel (B) is 100 µM. Imaging was carried out at 670 nm (red, Cy5), 520 nm (green, FITC) and 433 nm (blue, DAPI), respectively. Please click here to view a larger version of this figure.
Figure 3. MACS sorting of differentiated DE cell populations and representative staining after sorting. (A) CXCR4+ stained cells (Q1) are enriched after MACS sorting. The number of CXCR4- cells (Q4) is decreased. Depending on the cell line being used the differentiation protocol yields >80% CXCR4-positive cells8 and MACS may be used to further enrich them. (B) Undifferentiated cells express the pluripotency marker SOX2 (green) but not the DE marker FOXA2 (red). (C) After the MACS sorting only very few undifferentiated SOX2+ cells remain (red), whereas the sorted population almost uniformly expresses the DE marker FOXA2 (green). (B-C) Nuclei were counterstained with DAPI (blue). Scale bar in panel (C) is 100 µm and applies to all panels. Imaging was carried out at 670 nm (red, Cy5), 520 nm (green, FITC) and 433 nm (blue, DAPI), respectively. Please click here to view a larger version of this figure.
Currently used differentiation protocols rarely result in 100% differentiated cells. For reasons that still have to be addressed some cells resist the differentiation process. Depending on the efficiency of the used differentiation protocol and the propensity of the ESC line a certain number of residual pluripotent cells are commonly observed even after differentiation into the definitive endoderm. These residual cells may impair downstream differentiations or further analysis such as transcriptomics, proteomics, and miRNA expression analysis. Residual pluripotent cells or other unwanted lineages may also exhibit paracrine effects that may interfere with the differentiation goals. Consequently, the removal of these cells may result in improved reproducibility.
The purification of CXCR4+ expressing endoderm cells after differentiation can be used to enrich these populations and to remove cells that resisted the differentiation process. The surface DE marker protein CXCR4 can be used for the specific purification of endoderm cells. The MACS purification protocol at hand can be completed within less than 2 hr and can be executed in a simple bench top format in every cell culture lab without expensive laboratory equipment and devices. FACS purification is a commonly used technique but employs harsh conditions. During FACS purifications cells are usually kept in suspension for a prolonged period of time and the cells are subject to other challenging factors, e.g., high pressure in the nozzle of the FACS device. In comparison the MACS procedure is fast and gentle. This improves the cells' ability to reattach during reseeding after the purification process. To further ease this reattachment a ROCK inhibitor is added to the culture media after and prior to the purification to prevent apoptosis. Another important factor that influences reattachment is the density at which cells are reseeded. This strongly depends on the used cell line. The cell numbers used for seeding in this study are representative cell numbers that work well in our hands. However, there may be need to adjust these numbers for different cell lines. Both measures, the addition of a ROCK inhibitor and the evaluation of the cell numbers for reseeding, are critical to the success of the further cell culture after the sorting.
With the used DE differentiation protocol typically >70% CXCR4+ cells can be obtained without sorting8. The performance of the protocol used for DE generation consequentially influences the efficiency of the subsequent MACS purification protocol. In general, efficient differentiation (> 80% CXCR4+ cells) yields a higher purity after MACS sorting (up to 95%). Thus, the usage of this purification method reduces the number of undifferentiated cells substantially but 100% purity cannot be achieved. In the future, lineage specific surface markers may be defined that will permit the purification of more terminally differentiated cells. The MACS sorting procedure is the prime selection for this purpose.
The authors have nothing to disclose.
The skillful technical assistance of Jasmin Kresse is gratefully acknowledged.
Hues8 human embryonic stem cell line | Harvard Department of stem cell & regenerative biology | NA | Suitable cell line for endoderm generation |
Hes3 human embryonic stem cell line | ES Cell International | NA | Suitable and robust cell line for endoderm generation |
mTeSR1 | Stemcell Technologies | 5850 | ESC culture medium |
FCS | Biowest | S1860 | |
Advanced RPMI 1640 | Life Technologies | 12633012 | |
CD184 (CXCR4)-APC, human | Miltenyi Biotec | 130-098-357 | |
anti-APC MicroBeads | Miltenyi Biotec | 130-090-855 | |
OctoMACS Separator | Miltenyi Biotec | 130-042-109 | magnetic field |
Y-27632 | Selleck Chemicals | S1049 | ROCK inhibitor |
CHIR-99021 | Tocris Bioscience | 4423 | |
Activin A | Peprotech | 120-14 | |
Gentle Cell Dissociation Reagent | Stemcell Technologies | 7174 | Enzyme-free passaging solution, alternative: Trypsin/EDTA |
Matrigel* | Corning | 354277 | basement membrane matrix * solve and store in aliquots at -80 °C as outlined in the suppliers manual. Upon use, thaw on ice, dilute in 25 ml ice-cold knockout DMEM/F-12. Add 1 ml to each well of a 6-well plate and incubate for 45 min at room temperature. Remove the matrigel and use immediately. |
MS Columns | Miltenyi Biotec | 30-042-201 | |
MACS Separator | Miltenyi Biotec | 130-042-302 | |
Human FOXA2 FW gggagcggtgaagatgga |
Life Technologies | NA | |
Human FOXA2 REV tcatgttgctcacggaggagta |
Life Technologies | NA | |
Human GSC FW gaggagaaagtggaggtctggtt |
Life Technologies | NA | |
Human GSC REV ctctgatgaggaccgcttctg |
Life Technologies | NA | |
SOX17 TaqMan assay | Applied Biosystems | Hs00751752_s1 | |
Human SOX7 FW gatgctgggaaagtcgtggaagg |
Life Technologies | NA | |
Human SOX7 REV tgcgcggccggtacttgtag |
Life Technologies | NA | |
Human POU5F1 FW cttgctgcagaagtgggtggagg |
Life Technologies | NA | |
Human POU5F1 REV ctgcagtgtgggtttcgggca |
Life Technologies | NA | |
Human Nanog FW ccgagggcagacatcatcc |
Life Technologies | NA | |
Human Nanog REV ccatccactgccacatcttct |
Life Technologies | NA | |
Human TBP FW caa cag cct gcc acc tta cgc tc |
Life Technologies | NA | |
Human TBP REV agg ctg tgg ggt cag tcc agt g |
Life Technologies | NA | |
Human TUBA1A FW ggc agt gtt tgt aga ctt gga acc c |
Life Technologies | NA | |
Human TUBA1A REV tgt gat aag ttg ctc agg gtg gaa g |
Life Technologies | NA | |
Human G6PD FW agg ccg tca cca aga aca ttc a |
Life Technologies | NA | |
Human G6PD REV cga tga tgc ggt tcc agc cta t |
Life Technologies | NA | |
Anti-SOX2 | Santa Cruz Biotechnology | sc-17320 | |
Anti-FOXA2 | MerckMillipore | 07-633 | |
Anti-SOX17 | R&D Systems | AF1924 | |
NA = not applicable |