Conserved markers of fetal pancreatic epithelium permit prospective isolation of islet progenitor cells by FACS

Abstract

Prospective isolation and characterization of progenitor cells is a paradigmatic strategy for studies of organ development. However, extraction of viable cells, fractionation of lineages, and in vitro analysis of progenitors from the fetal pancreas in experimental organisms like mice has proved challenging and has not yet been reported for human fetal pancreas. Here, we report isolation of pancreatic islet progenitor cells from fetal mice by FACS. Monoclonal antibodies that recognize cell-surface proteins on candidate stem cells in brain, skin, and other organs enabled separation of major pancreatic cell lineages and isolation of native pancreatic cells expressing neurogenin 3, an established marker of islet progenitors. New in vitro cell culture methods permitted isolated mouse islet progenitors to develop into hormone-expressing endocrine cells. Insulin-producing cells derived in vitro required or expressed factors that regulate fetal beta cell differentiation; thus, the genetic programs normally controlling in vivo mouse islet development are similarly required in our system. Moreover, antibodies that recognize conserved orthologous cell-surface epitopes in human fetal pancreas allowed FACS-based enrichment of candidate islet progenitor cells expressing neurogenin 3. Our studies reveal previously undescribed strategies for prospective purification and analysis of pancreatic endocrine progenitor cells that should accelerate studies of islet development and replacement.

Fig. 1.

FACS-based isolation of NGN3⁺ endocrine progenitor cells from E15.5 mouse embryonic pancreas. (A–C) Immunohistology of E15.5 mouse pancreas. (A and B) NGN3⁺ cells are CD133⁺ (arrows). Images were taken by optical sectioning through multiple focal planes; representative images are shown. (C) Insulin⁺ cells are CD133^–. (D) FACS plot of sorted cells after exposure to anti-CD133 antibody. Percentages of CD133^– and CD133⁺ cells are indicated. (E) Subsets of isolated CD133^– cells express insulin and glucagon. Nuclei were visualized by DAPI staining (blue). (F) Subsets of CD133⁺ cells express NGN3. (G and H) Immunohistology showing expression of CD49f in E15.5 mouse pancreatic cells, including NGN3⁺ and insulin⁺ cells. (G) Magnified image of H. (I) FACS plot of cells sorted after exposure to anti-CD49f antibody. Percentages of CD49f^high, CD49f^low, and CD49f^– cells are indicated. (J) Subsets of isolated CD49f^−/low cells express insulin and glucagon. Cells expressing CarbA were not detected. (K) Subsets of isolated CD49f^high cells express CarbA (red). (L) FACS plot of cells sorted after exposure to antibodies recognizing CD133 and CD49f. Four fractions (labeled I–IV) are indicated. (M) CD49f^low CD133⁻ cells (fraction III) exclusively contain insulin⁺ or glucagon⁺ cells. (N) CD49f^low CD133⁺ cells (fraction II) exclusively contain NGN3⁺ cells. (E, J, and M) Insulin (white arrowheads); glucagon (green, arrows); (F and N) NGN3 (red, arrowheads).

Fig. 2.

In vitro differentiation of NGN3⁺ cells. (A) Schematic of the coculture strategy for assessing developmental potential of sorted fraction II single cells. (B–D) Expression of indicated β cell markers after coculture on PA6 feeders of fraction II cells from WT mice. (E and F) Coexpression of insulin and EGFP after coculture on PA6 feeders of fraction II cells from MIP-EGFP mice. (G) RT-PCR analysis of sorted cells before in vitro culture. (H) RT-PCR analysis of fraction II cells after coculture with MEFs for 2 days.

Fig. 3.

FACS and in vitro culture of E15.5 ngn3^–/– pancreatic cells. (A) FACS analysis of pancreatic cells from control (WT) and ngn3^–/– embryos revealed reduced cell numbers in fractions II and III (circled) from ngn3^–/– pancreas. (B) After 4 days of coculture, ngn3^–/– fraction II cells expressed detectable E-cadherin (green), an epithelial marker, but failed to produce detectable C-peptide (red).

Fig. 4.

Identification of a precursor cell population for NGN3⁺ cells. (A) Schematic of the coculture strategy for assessing developmental potential of sorted ngn3-EGFP^– fraction I single cells. (B) ngn3-EGFP⁺ cells (green) after 2 days of culture on an AC6 feeder layer. (C) Quantification of ngn3-EGFP⁺ cells during culture (n = 4). (D) RT-PCR analysis of ngn3 expression by fraction I cells before and after culture. Values represent mean ± SD.

Fig. 5.

Immunohistology and FACS of human fetal pancreas. (A and B) Immunohistology of human fetal pancreas at 14 weeks. NGN3⁺ cells express both CD49f (arrowheads) (A) and CD133 (arrows) (B). (C–E) FACS analysis of human fetal pancreas at 15 weeks. Dissociated cells were stained with antibodies recognizing CD133 (C and E) and CD49f (D and E). Four populations are outlined. (F) Quantitative RT-PCR analysis of ngn3 mRNA levels in cells after sorting of 15-week human fetal pancreas. ngn3 expression level was normalized to β-actin and shown in arbitrary unit. The average of two samples is shown. The pie chart shows the percentage of recovered cells in each indicated fraction.