Identification of a myofibroblast differentiation program during neonatal lung development.

Alveologenesis is the final stage of lung development in which the internal surface area of the lung is increased to facilitate efficient gas exchange in the mature organism. The first phase of alveologenesis involves the formation of septal ridges (secondary septae) and the second phase involves thinning of the alveolar septa. Within secondary septa, mesenchymal cells include a transient population of alveolar myofibroblasts (MyoFBs) and a stable but poorly described population of lipid-rich cells that have been referred to as lipofibroblasts or matrix fibroblasts (MatFBs). Using a unique Fgf18CreER lineage trace mouse line, cell sorting, single-cell RNA sequencing and primary cell culture, we have identified multiple subtypes of mesenchymal cells in the neonatal lung, including an immature progenitor cell that gives rise to mature MyoFB. We also show that the endogenous and targeted ROSA26 locus serves as a sensitive reporter for MyoFB maturation. These studies identify a MyoFB differentiation program that is distinct from other mesenchymal cell types and increases the known repertoire of mesenchymal cell types in the neonatal lung.

Identification of a myofibroblast differentiation program during neonatal lung development.

Alveologenesis is the final stage of lung development in which the internal surface area of the lung is increased to facilitate efficient gas exchange in the mature organism. The first phase of alveologenesis involves the formation of septal ridges (secondary septae) and the second phase involves thinning of the alveolar septa. Within secondary septa, mesenchymal cells include a transient population of alveolar myofibroblasts (MyoFB) and a stable but poorly described population of lipid rich cells that have been referred to as lipofibroblasts or matrix fibroblasts (MatFB). Using a unique Fgf18CreER lineage trace mouse line, cell sorting, single cell RNA sequencing, and primary cell culture, we have identified multiple subtypes of mesenchymal cells in the neonatal lung, including an immature progenitor cell that gives rise to mature MyoFB. We also show that the endogenous and targeted ROSA26 locus serves as a sensitive reporter for MyoFB maturation. These studies identify a myofibroblast differentiation program that is distinct form other mesenchymal cells types and increases the known repertoire of mesenchymal cell types in the neonatal lung.

Identification of a FGF18-expressing alveolar myofibroblast that is developmentally cleared during alveologenesis.

Alveologenesis is an essential developmental process that increases the surface area of the lung through the formation of septal ridges. In the mouse, septation occurs postnatally and is thought to require the alveolar myofibroblast (AMF). Though abundant during alveologenesis, markers for AMFs are minimally detected in the adult. After septation, the alveolar walls thin to allow efficient gas exchange. Both loss of AMFs or retention and differentiation into another cell type during septal thinning have been proposed. Using a novel Fgf18:CreERT2 allele to lineage trace AMFs, we demonstrate that most AMFs are developmentally cleared during alveologenesis. Lung mesenchyme also contains other poorly described cell types, including alveolar lipofibroblasts (ALF). We show that Gli1:CreERT2 marks both AMFs as well as ALFs, and lineage tracing shows that ALFs are retained in adult alveoli while AMFs are lost. We further show that multiple immune cell populations contain lineage-labeled particles, suggesting a phagocytic role in the clearance of AMFs. The demonstration that the AMF lineage is depleted during septal thinning through a phagocytic process provides a mechanism for the clearance of a transient developmental cell population.

Generation and validation of novel conditional flox and inducible Cre alleles targeting fibroblast growth factor 18 (Fgf18).

BACKGROUND: Fibroblast growth factor 18 (FGF18) functions in the development of several tissues, including the lung, limb bud, palate, skeleton, central nervous system, and hair follicle. Mice containing a germline knockout of Fgf18 (Fgf18 -/- ) die shortly after birth. Postnatally, FGF18 is being evaluated for pathogenic roles in fibrosis and several types of cancer. The specific cell types that express FGF18 have been difficult to identify, and the function of FGF18 in postnatal development and tissue homeostasis has been hampered by the perinatal lethality of Fgf18 null mice. RESULTS: We engineered a floxed allele of Fgf18 (Fgf18 flox ) that allows conditional gene inactivation and a CreERT2 knockin allele (Fgf18 CreERT2 ) that allows the precise identification of cells that express Fgf18 and their lineage. We validated the Fgf18 flox allele by targeting it in mesenchymal tissue and primary mesoderm during embryonic development, resulting in similar phenotypes to those observed in Fgf18 null mice. We also use the Fgf18 CreERT2 allele, in combination with a conditional fluorescent reporter to confirm known and identify new sites of Fgf18 expression. CONCLUSION: These alleles will be useful to investigate FGF18 function during organogenesis and tissue homeostasis, and to target specific cell lineages at embryonic and postnatal time points.

Use of dried plasma spots for the quantification of iothalamate in clinical studies.

BACKGROUND AND OBJECTIVES: Although iothalamate clearances have been widely used to measure GFR, the need for transportation of plasma samples under refrigerated conditions obviates its use in resource-poor situations. Spots of blood or plasma dried on filter paper may provide a solution. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Using a validated HPLC technique, iothalamate in dried blood spots of different hematocrits was measured. GFR was measured over 5 hours in 10 subjects with CKD using dried plasma spots and standard methods. RESULTS: Lower hematocrit produced greater area of blood spreading and lowered the recovery of iothalamate from dried blood spots. However, the relationship between iothalamate concentrations in dried plasma spots and plasma showed a regression slope of 0.95 (95% confidence interval=0.92-0.98, P<0.001). Bland-Altman plot of paired sample points (n=116) showed a bias of -4 µg/ml and limits of agreement of -38 to +30 µg/ml. The relationship between GFRs using dried plasma spots and plasma methods also showed an excellent relationship (slope of 0.95, 95% confidence interval=0.82-1.17). Bland-Altman plot of paired GFRs showed a bias of 2 ml/min, with limits of agreement of -6 to +10 ml/min. Precision was generally between 5% and 10%, and accuracy was within 5%. CONCLUSIONS: Although dried blood spots are unsuitable for studies among those patients with very low hematocrit, dried plasma spots correct for this limitation, and this small pilot study shows that it is a reasonably reliable method for quantifying iothalamate and subsequently, determining GFR.