Colony-forming cells in the adult mouse pancreas are expandable in Matrigel and form endocrine/acinar colonies in laminin hydrogel

Abstract

The study of hematopoietic colony-forming units using semisolid culture media has greatly advanced the knowledge of hematopoiesis. Here we report that similar methods can be used to study pancreatic colony-forming units. We have developed two pancreatic colony assays that enable quantitative and functional analyses of progenitor-like cells isolated from dissociated adult (2-4 mo old) murine pancreas. We find that a methylcellulose-based semisolid medium containing Matrigel allows growth of duct-like "Ring/Dense" colonies from a rare (∼1%) population of total pancreatic single cells. With the addition of roof plate-specific spondin 1, a wingless-int agonist, Ring/Dense colony-forming cells can be expanded more than 100,000-fold when serially dissociated and replated in the presence of Matrigel. When cells grown in Matrigel are then transferred to a Matrigel-free semisolid medium with a unique laminin-based hydrogel, some cells grow and differentiate into another type of colony, which we name "Endocrine/Acinar." These Endocrine/Acinar colonies are comprised mostly of endocrine- and acinar-like cells, as ascertained by RNA expression analysis, immunohistochemistry, and electron microscopy. Most Endocrine/Acinar colonies contain beta-like cells that secrete insulin/C-peptide in response to D-glucose and theophylline. These results demonstrate robust self-renewal and differentiation of adult Ring/Dense colony-forming units in vitro and suggest an approach to producing beta-like cells for cell replacement of type 1 diabetes. The methods described, which include microfluidic expression analysis of single cells and colonies, should also advance study of pancreas development and pancreatic progenitor cells.

Fig. 1.

“Ring” colonies are formed in Matrigel-containing culture from CD133⁺Sox9/EGFP⁺ cells isolated from dissociated adult murine pancreata. (A) A Ring colony starts as a “Small Bright” colony and grows into a Ring colony. (B) Flow cytometry analysis of CD133 and Sox9/EGFP expression of total dissociated adult pancreata. Regions (R) drawn indicate sorting windows. (C) PCFUs–Ring are most enriched in the CD133⁺Sox9/EGFP⁺ R3 window. (D) Single-colony microfluidic qRT-PCR analysis demonstrates that Small Bright and Ring colonies express high levels of ductal markers and low but detectable levels of endocrine and acinar cell markers. Each column is from a single colony. (E) Whole-mount and frozen section immunostaining demonstrated protein expression of ductal (Sox9, Mucin1, and Spp1), acinar (Amylase), or endocrine (C-Peptide) markers.

Fig. 2.

“Dense” colonies are induced by RSPO1 but not Dkk1 in Matrigel-containing culture and have enhanced progenitor cell marker expression compared with Ring colonies. (A) Representative photomicrographs of 3-wk-old colonies grown in the presence of designated factors. (B) Proportion of Dense colonies is increased by exogenous RSPO1 in a dose-dependent manner, with optimal dose at 750 ng/mL. (C) Colony-forming efficiency of CD133⁺Sox9/EGFP⁺ cells, determined by the total number of colonies formed from 2,500 input cells, was not changed by RSPO1 or Dkk1. (D) Microfluidic qRT-PCR analysis of individually handpicked colonies. Each bar is from a single colony. (E) Whole-mount immunostaining of a Dense colony.

Fig. 3.

“Endocrine/Acinar” colonies are formed from dissociated and replated Ring and Dense colonies in laminin hydrogel-containing culture. (A) Representative photomicrographs of 2-wk-old Endocrine/Acinar colonies. (B, Upper) Total 3-wk-old colonies grown in Matrigel-containing culture (stimulated without or with 750 ng/mL RSPO1) were dissociated and replated into laminin hydrogel colony assay for 2 wk. Colony-forming efficiency was calculated as the number of Endocrine/Acinar colonies generated divided by total number of input cells. (Lower) A total of 20 3-wk-old Ring (grown in Matrigel) or Dense colonies (grown in Matrigel and RSPO1) were picked, pooled, and dissociated into single-cell suspension. Total cell number was determined and the cells subsequently were replated into laminin hydrogel colony assay in quadruplicated wells for 2 wk. The conversion efficiency was calculated as percentage Endocrine/Acinar colony-forming efficiency times total cell number and then divided by 20. (C) Whole-mount immunostaining of Endocrine/Acinar colonies. (D) Transmission electronmicroscopy of Endocrine/Acinar colonies showing cells with insulin- (Upper) or acinar-like (Lower) granules. (E) Microfluidic qRT-PCR analysis of individually handpicked colonies. Each bar is from a single colony. (F) In vitro glucose change assay on pooled Endocrine/Acinar colonies. Concentrations of theophylline are 10 mM.

Fig. 4.

RSPO1, but not Dkk1, supports expansion of PCFUs–Ring/Dense long term in vitro. As illustrated in A, CD133⁺Sox9/EGFP⁺ cells were plated in culture containing Matrigel and RSPO1. Three weeks later, colonies were counted and total colonies from each well (n = 4 for each group) were procured, dissociated, and a fraction of total cells was replated in the presence of Matrigel and designated factors. After 2 wk, the colonies were counted and all cells were procured, dissociated, counted, and replated. Counting, procurement, dissociation, and replating procedure was repeated a total of four times. (B) The number of total resulting colonies (adjusted for fractions of plated cells) was analyzed. Data represent two experiments with similar trends. *P < 0.05 compared with vehicle control. (C) Morphological change of colonies toward smaller Dense colonies with increased passages was noted in cultures continuously receiving exogenous RSPO1.

Fig. 5.

Model of PCFU–Ring/Dense expansion and differentiation.