PVLSI (Pioneer Valley Life Sciences Institute) Posters - 2019

PVLSI (Pioneer Valley Life Sciences Institute) Posters - 2019https://scholarlycommons.libraryinfo.bhs.org/research_education/1014/thumbnail.jp

Reduced Satellite Cell Numbers and Myogenic Capacity in Aging Can Be Alleviated by Endurance Exercise

Background: Muscle regeneration depends on satellite cells, myogenic stem cells that reside on the myofiber surface. Reduced numbers and/or decreased myogenic aptitude of these cells may impede proper maintenance and contribute to the age-associated decline in muscle mass and repair capacity. Endurance exercise was shown to improve muscle performance; however, the direct impact on satellite cells in aging was not yet thoroughly determined. Here, we focused on characterizing the effect of moderate-intensity endurance exercise on satellite cell, as possible means to attenuate adverse effects of aging. Young and old rats of both genders underwent 13 weeks of treadmill-running or remained sedentary. Methodology: Gastrocnemius muscles were assessed for the effect of age, gender and exercise on satellite-cell numbers and myogenic capacity. Satellite cells were identified in freshly isolated myofibers based on Pax7 immunostaining (i.e., exvivo). The capacity of individual myofiber-associated cells to produce myogenic progeny was determined in clonal assays (in-vitro). We show an age-associated decrease in satellite-cell numbers and in the percent of myogenic clones in old sedentary rats. Upon exercise, there was an increase in myofibers that contain higher numbers of satellite cells in both young and old rats, and an increase in the percent of myogenic clones derived from old rats. Changes at the satellite cell level in old rats were accompanied with positive effects on the lean-to-fat Gast muscle composition and on spontaneous locomotion levels. The significance of these data is that they suggest that the endurance exercise-mediated boost in bot

Cell Hierarchy and Lineage Commitment in the Bovine Mammary Gland

The bovine mammary gland is a favorable organ for studying mammary cell hierarchy due to its robust milk-production capabilities that reflect the adaptation of its cell populations to extensive expansion and differentiation. It also shares basic characteristics with the human breast, and identification of its cell composition may broaden our understanding of the diversity in cell hierarchy among mammals. Here, Lin− epithelial cells were sorted according to expression of CD24 and CD49f into four populations: CD24medCD49fpos (putative stem cells, puStm), CD24negCD49fpos (Basal), CD24highCD49fneg (putative progenitors, puPgt) and CD24medCD49fneg (luminal, Lum). These populations maintained differential gene expression of lineage markers and markers of stem cells and luminal progenitors. Of note was the high expression of Stat5a in the puPgt cells, and of Notch1, Delta1, Jagged1 and Hey1 in the puStm and Basal populations. Cultured puStm and Basal cells formed lineage-restricted basal or luminal clones and after re-sorting, colonies that preserved a duct-like alignment of epithelial layers. In contrast, puPgt and Lum cells generated only luminal clones and unorganized colonies. Under non-adherent culture conditions, the puPgt and puStm populations generated significantly more floating colonies. The increase in cell number during culture provides a measure of propagation potential, which was highest for the puStm cells. Taken together, these analyses position puStm cells at the top of the cell hierarchy and denote the presence of both bi-potent and luminally restricted progenitors. In addition, a population of differentiated luminal cells was marked. Finally, combining ALDH activity with cell-surface marker analyses defined a small subpopulation that is potentially stem cell- enriched

<i>In-situ</i> localization of proteins with distinct expression among the bMEC populations.

<p>All analyses depict immunofluorescence detection, except for ALDH1 which was detected by DAB reaction, generating a brown signal with hematoxylin counterstaining of the nuclei. ALDH1: red arrows mark positively stained cells in the stromal area. Notch1: red arrows mark positively stained cells in the basal layer. Bar = 50 µm.</p

Proposed bovine mammary epithelial cell hierarchy.

<p>Proposed bovine mammary epithelial cell hierarchy.</p

NSFC development and characteristics depend on its origin.

<p>A: Representative demonstration of limited development of a cultured NSFC from Lum cells compared to NSFCs from the other populations. B: Serial dissociation and culture of NSFCs over three generations demonstrates limited self-renewal capacity and a more severe effect of sorting, compared with antibody labeling, on their development. C: puStm and puPgt cultures generate higher numbers of NSFCs compared with Basal and Lum cultures. Columns represent mean±SEM of three analyses of least 26 floating colonies for each population. Different letters above the columns indicate statistically significant (<i>P</i><0.05) differences. Bar = 50 µm.</p

Incorporating ALDH activity into the CD24/CD49f-based analysis reveals a small ALDH^br population within the puStm fraction that is potentially enriched with stem cells.

<p>A: FACS analysis of Lin⁻ bMECs and gating of ALDH-positive (ALDH^br) cells according to the effect of the ALDH inhibitor DEAB. B: Demonstration of ALDH^br (red) and ALDH^neg (green) distribution among the populations sorted according to CD49f and CD24 expression. SSC - side scatter.</p

Luminal and basal/myoepithelial layers in the bovine mammary gland can be distinguished by immunofluorescence analysis.

<p>A: H&E staining of sections from a heifer's mammary gland reveals ductal structures penetrating the fibrous stroma. B–F: Immunofluorescence detection of mammary lineage markers. Inset: 2× magnification. L = lumen. Bar = 50 µm.</p

Four populations of epithelial cells with distinct CD24 and CD49f expression were identified in the bovine mammary gland.

<p>Lin⁻ bMECs from heifer mammary gland were sorted according to CD24 and CD49f expression. Two main populations: CD24^neg-medCD49f^pos and CD24^med-highCD49f^neg, encircled by dashed green lines, emerged in the density plot. Putative populations enriched with stem cells (puStm, 5.8±1.3%) and their progenitors (puPgt, 8.0±1.4%), as well as their complementary Basal (11.7±2.9%) and luminal (Lum, 12.2±2.6%) populations (encircled by solid red lines) were collected. Inset: gating of living cells (framed in red) according to PI staining. Percentage of each population was calculated out of the total living cells detected. FSC – forward scatter.</p

Cultured bMEC populations do not differ in their CD49f/CD24 expression, but maintain their distinct parental characteristics.

<p>A: Schematic representation of the experimental procedure. B: FACS histograms depicting the levels of CD24 and CD49f in freshly isolated bMECs compared with their cultured counterparts. C. FACS dot-plots depicting the subpopulations sorted from the cultured cells. D: Immunofluorescence staining of the lineage markers CK14 and CK18 in organized and non-organized colonies. E: Regardless their different cell-surface marker expression, organized colonies were significantly more frequent in sorted cultured cells originated from the puStm and Basal populations. Bar = 50 µm.</p