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Identification of a proximal progenitor population from murine fetal lungs with clonogenic and multilineage differentiation potential.

ABSTRACT: Lung development-associated diseases are major causes of morbidity and lethality in preterm infants and children. Access to the lung progenitor/stem cell populations controlling pulmonary development during embryogenesis and early postnatal years is essential to understand the molecular basis of such diseases. Using a Nkx2-1(mCherry) reporter mouse, we have identified and captured Nkx2-1-expressing lung progenitor cells from the proximal lung epithelium during fetal development. These cells formed clonal spheres in semisolid culture that could be maintained in vitro and demonstrated self-renewal and expansion capabilities over multiple passages. In-vitro-derived Nkx2-1-expressing clonal spheres differentiated into a polarized epithelium comprised of multiple cell lineages, including basal and secretory cells, that could repopulate decellularized lung scaffolds. Nkx2-1 expression thus defines a fetal lung epithelial progenitor cell population that can be used as a model system to study pulmonary development and associated pediatric diseases.

SUBMITTER: Bilodeau M

PROVIDER: S-EPMC4223706 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Identification of a proximal progenitor population from murine fetal lungs with clonogenic and multilineage differentiation potential.

Bilodeau Mélanie M Shojaie Sharareh S Ackerley Cameron C Post Martin M Rossant Janet J

Stem cell reports 20140904 4

Lung development-associated diseases are major causes of morbidity and lethality in preterm infants and children. Access to the lung progenitor/stem cell populations controlling pulmonary development during embryogenesis and early postnatal years is essential to understand the molecular basis of such diseases. Using a Nkx2-1(mCherry) reporter mouse, we have identified and captured Nkx2-1-expressing lung progenitor cells from the proximal lung epithelium during fetal development. These cells form ...[more]

PMID: 25358791

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Project description:BackgroundHuman induced pluripotent stem cells (hiPSCs) offer a renewable source of cells for the generation of hematopoietic cells for cell-based therapy, disease modeling, and drug screening. However, current serum/feeder-free differentiation protocols rely on the use of various cytokines, which makes the process very costly or the generation of embryoid bodies (EBs), which are labor-intensive and can cause heterogeneity during differentiation. Here, we report a simple feeder and serum-free monolayer protocol for efficient generation of iPSC-derived multipotent hematoendothelial progenitors (HEPs), which can further differentiate into endothelial and hematopoietic cells including erythroid and T lineages.MethodsFormation of HEPs from iPSCs was initiated by inhibition of GSK3 signaling for 2 days followed by the addition of VEGF and FGF2 for 3 days. The HEPs were further induced toward mature endothelial cells (ECs) in an angiogenic condition and toward T cells by co-culturing with OP9-DL1 feeder cells. Endothelial-to-hematopoietic transition (EHT) of the HEPs was further promoted by supplementation with the TGF-β signaling inhibitor. Erythroid differentiation was performed by culturing the hematopoietic stem/progenitor cells (HSPCs) in a three-stage erythroid liquid culture system.ResultsOur protocol significantly enhanced the number of KDR+ CD34+ CD31+ HEPs on day 5 of differentiation. Further culture of HEPs in angiogenic conditions promoted the formation of mature ECs, which expressed CD34, CD31, CD144, vWF, and ICAM-1, and could exhibit the formation of vascular-like network and acetylated low-density lipoprotein (Ac-LDL) uptake. In addition, the HEPs were differentiated into CD8+ T lymphocytes, which could be expanded up to 34-fold upon TCR stimulation. Inhibition of TGF-β signaling at the HEP stage promoted EHT and yielded a large number of HSPCs expressing CD34 and CD43. Upon erythroid differentiation, these HSPCs were expanded up to 40-fold and displayed morphological changes following stages of erythroid development.ConclusionThis protocol offers an efficient and simple approach for the generation of multipotent HEPs and could be adapted to generate desired blood cells in large numbers for applications in basic research including developmental study, disease modeling, and drug screening as well as in regenerative medicine.

Dataset Information

Identification of a proximal progenitor population from murine fetal lungs with clonogenic and multilineage differentiation potential.

Publications

Identification of a proximal progenitor population from murine fetal lungs with clonogenic and multilineage differentiation potential.

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