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A Critical Re-Evaluation of CD24-Positivity of Human Embryonic Stem Cells Differentiated into Pancreatic Progenitors

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Abstract

Differentiation of embryonic stem cells (ESCs) into insulin-producing cells for cell replacement therapy of diabetes mellitus comprises the stepwise recapitulation of in vivo developmental stages of pancreatic organogenesis in an in vitro differentiation protocol. The chemical compounds IDE-1 and (-)-indolactam-V can be used to direct mouse and human ESCs through these stages to form definitive endoderm via an intermediate mesendodermal stage and finally into pancreatic endoderm. Cells of the pancreatic endoderm express the PDX1 transcription factor and contribute to all pancreatic cell types upon further in vitro or in vivo differentiation. Even though this differentiation approach is highly effective and reproducible, it generates heterogeneous populations containing PDX1-expressing pancreatic progenitors amongst other cell types. Thus, a technique to separate PDX1-expressing cells from this mixture is very desirable. Recently it has been reported that PDX1-positive pancreatic progenitors, derived from human embryonic stem cells, express the surface marker CD24. Therefore were subjected mouse and human ESCs to a small molecule differentiation approach and the expression of the surface marker CD24 was monitored in undifferentiated cells, cells committed to the definitive endoderm and cells reminiscent of the pancreatic endoderm. We observed that both mouse and human ESCs expressed CD24 in the pluripotent state, during the whole process of endoderm formation and upon further differentiation towards pancreatic endoderm. Thus CD24 is not a suitable cell surface marker for identification of PDX1-positive progenitor cells.

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Acknowledgments

This work has been supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) within the framework of the Cluster of Excellence REBIRTH (From Regenerative Biology to Reconstructive Therapy).

Conflict of interest

The authors declare no potential conflicts of interest.

Author information

Authors and Affiliations

  1. Institute of Clinical Biochemistry, Hannover Medical School, 30625, Hannover, Germany

    Ortwin Naujok & Sigurd Lenzen

Authors
  1. Ortwin Naujok
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  2. Sigurd Lenzen
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Corresponding author

Correspondence to Sigurd Lenzen.

Additional information

This work was supported by the Deutsche Forschungsgemeinschaft (German Research Foundation) within the framework of the Cluster of Excellence REBIRTH.

Electronic Supplementary Material

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Supplemental Table 1

qPCR primer (DOC 71 kb)

Supplemental Table 2

Binding of control antibodies to undifferentiated and differentiated ES cells at the D0, D6 and D10 time points. (DOC 31 kb)

Supplemental Figure 1

Comparison of the staining pattern of two different anti-CD24 antibodies. (A) Immunocytochemical staining of CD24/NANOG (CD24 unconjugated, clone SN3, Santa Cruz; Anti-Nanog, Abcam, Cambridge, UK) in undifferentiated hESCs (d0). (B) Immunocytochemical staining of CD24/NANOG (CD24 unconjugated, clone Ml5, Biolegend; Anti-NANOG, Abcam, Cambridge, UK) in undifferentiated hESCs (d0). The staining of both antibodies showed mosaic-like, aggregated plasma membrane localization. Images taken were post processed using Autoquant X (Media Cybernetics, Bethesda, USA) and Imaris (Bitplane, Zürich, Switzerland). Original magnification 400x. (DOC 12,982 kb)

Supplemental Figure 2

Comparison of the staining pattern of two different anti-CD24 antibodies at day 6 and day 10 of differentiation. (A) Immunocytochemical triple-staining of CD24/SOX17/FOXA2 using both anti-CD24 antibodies from Biolegend (clone ML5) and Santa Cruz (clone SN3). Shown are cells hESCs after 6 of differentiation with IDE-1 and sodium butyrate. The cells were double-positive for SOX17 and FOXA2, which marks cells of the definitive endoderm. In addition these cells were positive for CD24. An area with non-DE cells is marked also positive for this marker. (B) Immunocytochemical double staining for CD24 and PDX1 using both anti-CD24 antibodies from Biolegend and Santa Cruz. Arrows indicate pdx1-negative cells; arrowheads indicate PDX1-positive cells. The staining of both antibodies showed mosaic-like, aggregated plasma membrane localization. Original magnification 400×. (DOC 24,688 kb)

Supplemental Figure 3

Flow cytometric analysis of human and mouse peripheral blood lymphocytes (A) Flow cytometry of gated human lymphocytes after staining with primary conjugated antibodies against the B-lymphocyte surface markers CD19 and CD24. The clone ml5 from Biolegend was used in this experimental setup. (B) Flow cytometry of gated NMRI mouse lymphocytes after staining with primary conjugated antibodies against the B-lymphocyte surface marker genes CD19/CD24 and CD45R/CD24. The numbers in the lower right of each dotplot depicts cell percentages of cells positive for one or both respective markers in each quadrant. (DOC 212 kb)

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Naujok, O., Lenzen, S. A Critical Re-Evaluation of CD24-Positivity of Human Embryonic Stem Cells Differentiated into Pancreatic Progenitors. Stem Cell Rev and Rep 8, 779–791 (2012). https://doi.org/10.1007/s12015-012-9362-y

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