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Acellular lung scaffolds direct differentiation of endoderm to functional airway epithelial cells: requirement of matrix-bound HS proteoglycans.

Shojaie S, Ermini L, Ackerley C, Wang J, Chin S, Yeganeh B, Bilodeau M, Sambi M, Rogers I, Rossant J, Bear CE, Post M - Stem Cell Reports (2015)

Bottom Line: Here we examined the role of the lung ECM in differentiation of pluripotent cells in vitro and demonstrate the robust inductive capacity of the native lung matrix alone.Extended culture of stem cell-derived definitive endoderm on decellularized lung scaffolds in defined, serum-free medium resulted in differentiation into mature airway epithelia, complete with ciliated cells, club cells, and basal cells with morphological and functional similarities to native airways.Heparitinase I, but not chondroitinase ABC, treatment of scaffolds revealed that the differentiation achieved is dependent on heparan sulfate proteoglycans and its bound factors remaining on decellularized scaffolds.

View Article: PubMed Central - PubMed

Affiliation: Program in Physiology and Experimental Medicine, Peter Gilgan Centre for Research and Learning, Hospital for Sick Children, Toronto, ON M5G 0A4, Canada; Department of Physiology, Faculty of Medicine, University of Toronto, Toronto, ON M5S1A8, Canada.

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Seeded Endodermal Cells Differentiate to NKX2-1+/SOX2+ Proximal Lung Progenitors with Culture on Decellularized Scaffolds(A) Schematic representation of differentiation to lung progenitor cells.(B and C) Real-time PCR analysis reveals an upregulation of Foxa2 and lung progenitor marker Nkx2-1 expression. Expression is presented relative to E13.5 mouse lungs, mean ± SEM, n = 4 experiments.(D and E) Proximal airway progenitor marker Sox2 and distal alveolar progenitor marker Sox9 were both detected, n = 4 experiments; with extended culture (day 21), Sox2 expression is upregulated.(F and G) NKX2-1mcherry+ cells are quantified with flow cytometry. The proportion of NKX2-1+ cells increases with extended culture at day 21 to 46.42% ± 3.6%, n = 3 experiments.(H and I) Immunostaining of day 7 cultures shows the presence of NKX2-1+/SOX2+/TRP63+ coexpressing airway basal stem cells. A small population of distal lineage NKX2-1+/SOX9+ progenitor cells is also present at this time point. (H) Scale bar represents 13 μm; (I) scale bar represents 25 μm.(J and K) Immunostaining of day 21 cultures for NKX2-1, SOX2, and SOX9 reveals that the majority of NKX2-1+ cells coexpress SOX2, while SOX9 protein expression is rare. Scale bar represents 50 μm.See also Figures S1 and S2.
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fig1: Seeded Endodermal Cells Differentiate to NKX2-1+/SOX2+ Proximal Lung Progenitors with Culture on Decellularized Scaffolds(A) Schematic representation of differentiation to lung progenitor cells.(B and C) Real-time PCR analysis reveals an upregulation of Foxa2 and lung progenitor marker Nkx2-1 expression. Expression is presented relative to E13.5 mouse lungs, mean ± SEM, n = 4 experiments.(D and E) Proximal airway progenitor marker Sox2 and distal alveolar progenitor marker Sox9 were both detected, n = 4 experiments; with extended culture (day 21), Sox2 expression is upregulated.(F and G) NKX2-1mcherry+ cells are quantified with flow cytometry. The proportion of NKX2-1+ cells increases with extended culture at day 21 to 46.42% ± 3.6%, n = 3 experiments.(H and I) Immunostaining of day 7 cultures shows the presence of NKX2-1+/SOX2+/TRP63+ coexpressing airway basal stem cells. A small population of distal lineage NKX2-1+/SOX9+ progenitor cells is also present at this time point. (H) Scale bar represents 13 μm; (I) scale bar represents 25 μm.(J and K) Immunostaining of day 21 cultures for NKX2-1, SOX2, and SOX9 reveals that the majority of NKX2-1+ cells coexpress SOX2, while SOX9 protein expression is rare. Scale bar represents 50 μm.See also Figures S1 and S2.

Mentions: During embryonic development, lung-specific endoderm progenitors originate from definitive anterior endoderm found in the developing foregut (Murry and Keller, 2008; Zorn and Wells, 2009). Therefore, we first generated definitive endoderm from mouse embryonic stem cells (ESCs) using activin A (Gouon-Evans et al., 2006; Kubo et al., 2004) and isolated an enriched population of endodermal cells by fluorescence-activated cell sorting for coexpression of CXCR4 and cKIT (Figures S2A and S2B). Sorted cells were seeded onto 350 μm thick sections of decellularized scaffolds and cultured in a supportive base media for up to 3 weeks without the addition of exogenous factors. To better recapitulate the lung microenvironment, we maintained cell-matrix constructs under air-liquid interface (ALI) culture conditions (Figure S2C). By 7 days of culture, seeded endodermal cells presented a pattern of organization reminiscent of the developing lung, lined by basement membrane proteins collagen IV and laminin (Figures S2D and S2E). Tubule structures were formed, and over half of the seeded population coexpressed pan-epithelial cell markers CDH1 and panKRT (Figure S2F). RT-PCR analysis showed maintenance of endoderm transcription factor Foxa2 expression for the duration of culture on scaffolds (Figure 1B). Nkx2-1 is an important transcriptional regulator of the lung that is one of the earliest markers for emergence of lung-specific endodermal cells (Kimura et al., 1996; Minoo et al., 1999). There was upregulation of Nkx2-1 after 7 days of culture on scaffolds, and expression was maintained for up to 21 days (Figure 1C); proximal (Sox2) and distal (Sox9) epithelial progenitor markers were both detected at the mRNA level, although Sox2 levels were greater (Figures 1D and 1E).


Acellular lung scaffolds direct differentiation of endoderm to functional airway epithelial cells: requirement of matrix-bound HS proteoglycans.

Shojaie S, Ermini L, Ackerley C, Wang J, Chin S, Yeganeh B, Bilodeau M, Sambi M, Rogers I, Rossant J, Bear CE, Post M - Stem Cell Reports (2015)

Seeded Endodermal Cells Differentiate to NKX2-1+/SOX2+ Proximal Lung Progenitors with Culture on Decellularized Scaffolds(A) Schematic representation of differentiation to lung progenitor cells.(B and C) Real-time PCR analysis reveals an upregulation of Foxa2 and lung progenitor marker Nkx2-1 expression. Expression is presented relative to E13.5 mouse lungs, mean ± SEM, n = 4 experiments.(D and E) Proximal airway progenitor marker Sox2 and distal alveolar progenitor marker Sox9 were both detected, n = 4 experiments; with extended culture (day 21), Sox2 expression is upregulated.(F and G) NKX2-1mcherry+ cells are quantified with flow cytometry. The proportion of NKX2-1+ cells increases with extended culture at day 21 to 46.42% ± 3.6%, n = 3 experiments.(H and I) Immunostaining of day 7 cultures shows the presence of NKX2-1+/SOX2+/TRP63+ coexpressing airway basal stem cells. A small population of distal lineage NKX2-1+/SOX9+ progenitor cells is also present at this time point. (H) Scale bar represents 13 μm; (I) scale bar represents 25 μm.(J and K) Immunostaining of day 21 cultures for NKX2-1, SOX2, and SOX9 reveals that the majority of NKX2-1+ cells coexpress SOX2, while SOX9 protein expression is rare. Scale bar represents 50 μm.See also Figures S1 and S2.
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fig1: Seeded Endodermal Cells Differentiate to NKX2-1+/SOX2+ Proximal Lung Progenitors with Culture on Decellularized Scaffolds(A) Schematic representation of differentiation to lung progenitor cells.(B and C) Real-time PCR analysis reveals an upregulation of Foxa2 and lung progenitor marker Nkx2-1 expression. Expression is presented relative to E13.5 mouse lungs, mean ± SEM, n = 4 experiments.(D and E) Proximal airway progenitor marker Sox2 and distal alveolar progenitor marker Sox9 were both detected, n = 4 experiments; with extended culture (day 21), Sox2 expression is upregulated.(F and G) NKX2-1mcherry+ cells are quantified with flow cytometry. The proportion of NKX2-1+ cells increases with extended culture at day 21 to 46.42% ± 3.6%, n = 3 experiments.(H and I) Immunostaining of day 7 cultures shows the presence of NKX2-1+/SOX2+/TRP63+ coexpressing airway basal stem cells. A small population of distal lineage NKX2-1+/SOX9+ progenitor cells is also present at this time point. (H) Scale bar represents 13 μm; (I) scale bar represents 25 μm.(J and K) Immunostaining of day 21 cultures for NKX2-1, SOX2, and SOX9 reveals that the majority of NKX2-1+ cells coexpress SOX2, while SOX9 protein expression is rare. Scale bar represents 50 μm.See also Figures S1 and S2.
Mentions: During embryonic development, lung-specific endoderm progenitors originate from definitive anterior endoderm found in the developing foregut (Murry and Keller, 2008; Zorn and Wells, 2009). Therefore, we first generated definitive endoderm from mouse embryonic stem cells (ESCs) using activin A (Gouon-Evans et al., 2006; Kubo et al., 2004) and isolated an enriched population of endodermal cells by fluorescence-activated cell sorting for coexpression of CXCR4 and cKIT (Figures S2A and S2B). Sorted cells were seeded onto 350 μm thick sections of decellularized scaffolds and cultured in a supportive base media for up to 3 weeks without the addition of exogenous factors. To better recapitulate the lung microenvironment, we maintained cell-matrix constructs under air-liquid interface (ALI) culture conditions (Figure S2C). By 7 days of culture, seeded endodermal cells presented a pattern of organization reminiscent of the developing lung, lined by basement membrane proteins collagen IV and laminin (Figures S2D and S2E). Tubule structures were formed, and over half of the seeded population coexpressed pan-epithelial cell markers CDH1 and panKRT (Figure S2F). RT-PCR analysis showed maintenance of endoderm transcription factor Foxa2 expression for the duration of culture on scaffolds (Figure 1B). Nkx2-1 is an important transcriptional regulator of the lung that is one of the earliest markers for emergence of lung-specific endodermal cells (Kimura et al., 1996; Minoo et al., 1999). There was upregulation of Nkx2-1 after 7 days of culture on scaffolds, and expression was maintained for up to 21 days (Figure 1C); proximal (Sox2) and distal (Sox9) epithelial progenitor markers were both detected at the mRNA level, although Sox2 levels were greater (Figures 1D and 1E).

Bottom Line: Here we examined the role of the lung ECM in differentiation of pluripotent cells in vitro and demonstrate the robust inductive capacity of the native lung matrix alone.Extended culture of stem cell-derived definitive endoderm on decellularized lung scaffolds in defined, serum-free medium resulted in differentiation into mature airway epithelia, complete with ciliated cells, club cells, and basal cells with morphological and functional similarities to native airways.Heparitinase I, but not chondroitinase ABC, treatment of scaffolds revealed that the differentiation achieved is dependent on heparan sulfate proteoglycans and its bound factors remaining on decellularized scaffolds.

View Article: PubMed Central - PubMed

Affiliation: Program in Physiology and Experimental Medicine, Peter Gilgan Centre for Research and Learning, Hospital for Sick Children, Toronto, ON M5G 0A4, Canada; Department of Physiology, Faculty of Medicine, University of Toronto, Toronto, ON M5S1A8, Canada.

Show MeSH
Related in: MedlinePlus