Characterization of murine mammary stem/progenitor cells in a D-galactose-induced aging model

D-galactose induces elevated inflammatory signature

The mice treated with D-galactose exhibited significantly higher plasma levels of inflammatory cytokine IL-6 and TNFα than the control group (Figure 1A). Specifically, the levels of IL-6 and TNFα were increased 1.6- and 1.9-fold, respectively. Mammary glands from D-galactose-treated mice had enlarged lymph nodes, which on average increased approximately 1.45-fold when measured by area (Figure 1B). In addition, p-STAT5, a downstream regulator of IL-6 and TNFα, was detected at a higher frequency of dark staining in mammary glands from D-galactose-treated mice (83%) than those from control mice (64%) (Figure 1C). The D-galactose group also demonstrated a significantly lower level of p-STAT5 light staining than the control (16% vs. 31%) (Figure 1C).

Figure 1. D-galactose induces elevated inflammatory signature. (A) Levels of inflammatory cytokine IL-6 and TNFα in serum samples collected from control mice and those treated with D-galactose (n = 3); (B) The area of lymph nodes in the mammary glands from control and D-galactose-treated mice (n = 5); (C) Quantification of p-STAT5 staining in mammary glands from control and D-galactose-treated mice (n = 4). Scale bars, 50 μm. Asterisks, significant difference between control and D-galactose (^*P < .05, ^**P < .01).

D-galactose decreases branching point and increases hyperplasia in mammary glands

Mice treated with D-galactose showed altered mammary glands that were characterized by decreased branching points and higher frequency of hyperplastic mammary ducts (Figure 2). The branching points per millimeter (mm) mammary duct were decreased, with 4.9 ± 1.5 in control mice and 2.7 ± 0.9 in D-galactose-treated mice (P < 0.05) (Figure 2A, 2C). The frequency of hyperplastic duct increased from 21.9 ± 4.9% in control mice to 42.8 ± 10.1% in D-galactose-treated mice (P < .01) (Figure 2B, 2C).

Figure 2. D-galactose affects mammary gland morphology and pathology. (A) Representative examples of whole mount carmine alum staining of mammary glands from control and D-galactose-treated mice (scale bars, 1 mm); (B) H&E histological images showing normal and hyperplastic ducts from control and D-galactose-treated mice (scale bars, 100 μm); (C) Quantification of branch points per millimeter (mm) duct (upper panel) and percent of hyperplastic ducts (lower panel) in control and D-galactose-treated mice (n = 5). Asterisks, significant difference between control and D-galactose (^*P < .05, ^**P < .01).

D-galactose alters mammary basal/luminal cell pools

Mammary epithelial cells are composed of basal and luminal cells. Flow cytometry analysis revealed increased basal cells (CD24^lowCD49^high) and decreased luminal cells (CD24^highCD49^low) in mammary glands harvested from D-galactose-treated mice when compared to control mice (Figure 3A). Quantitative analysis showed that the frequency of basal cells increased from 14.8 ± 2.4% in the control mice to 25.5 ± 8.7% in the D-galactose-treated mice (Figure 3B). In contrast, the frequency of luminal cells decreased from 38.2 ± 7.9% in control mice to 23.5 ± 5.1% in D-galactose-treated mice (Figure 3B). As a consequence, the luminal-to-basal cell ratio decreased from 2.6 ± 0.5 in control mice to 1.0 ± 0.2 in D-galactose-treated mice (Figure 3B).

Figure 3. D-galactose alters mammary basal/luminal cell pools. (A) Representative flow cytometry analysis of mammary epithelial cells from control and D-galactose-treated mice. Basal cells express high levels of CD49f, and luminal cells express high levels of CD24; (B) Quantification of % basal cell, % luminal cell, and luminal-to-basal cell ratio in mammary epithelial cells isolated from control and D-galactose-treated mice (n = 5). Asterisks, significant difference between control and D-galactose (^*P < .05).

D-galactose alters stem/progenitor cell function in vitro

Mammary epithelium is orchestrated by a hierarchy of multipotent and unipotent stem cells; however, it is generally agreed that unipotent basal and luminal stem cells are responsible for routine epithelial maintenance [21, 22]. In this study, we refer to stem cells enriched in the basal cell fraction as mammary stem cells (MaSCs) and those enriched in the luminal cell fraction as luminal progenitors (LPs) to be consistent with existing literature. Both MaSCs and LPs were assessed for their in vitro self-renewal function using the 3D organoid serial passage assay. To obtain 3D organoids, sorted basal or luminal cells were first allowed to form spheres in suspension culture and then transferred to 3D Matrigel culture for sphere differentiation. The 3D organoids formed by spheres exhibited distinct morphological differences between MaSCs and LPs, with the former forming solid structures and the latter forming hollow-like structures [23]. These individual 3D organoids were dissociated into a single cell suspension and passaged in vitro for five generations (Figure 4A, 4B). For MaSCs, out of 30 basal 3D organoids in the control mice, 25 of them (83%) were able to be passaged to P5, while only 27% of basal 3D organoids from D-galactose-treated mice successfully reached P5 (Figure 4C), indicating declined self-renewal function. Similarly, for LPs, the number of 3D organoids that could be passaged to P5 decreased from 50% in the control mice to 10% in the D-galactose-treated mice (Figure 4C).

Figure 4. D-galactose alters mammary stem/progenitor cell function in vitro. (A) Representative images showing in vitro serial passage of basal stem cell contained 3D organoids from primary organoids (P0) to P1 and P5 (scale bars, 100 μm); (B) Representative images showing in vitro serial passage of luminal progenitor cell contained 3D organoids from primary organoids (P0) to P1 and P5 (scale bars, 100 μm); (C) Quantification of the number of 3D organoids that can be passaged to P5 for stem/progenitor cells derived from control and D-galactose-treated mice (for each type of 3D organoids, 10 organoids per animal × 3 animals = 30 organoids were assayed).

We also characterized LP differentiation in vitro using the 2D colony forming assay, where LPs were found to form morphologically distinct colonies when cultured on plates pre-seeded with irradiated NIH-3T3 fibroblasts (Figure 5A). Colonies formed by LPs from D-galactose-treated mice were dominated by K8⁺K14⁺ bipotent colonies (55 ± 9%), while those from control mice were dominated by K8⁺K14^– colonies (43 ± 10%) (Figure 5B). In fact, there was a significant reduction of K8⁺K14^– colonies (24 ± 6%) in D-galactose-treated mice. The percentage of colonies displaying K8^–K14⁺ was similar between control (24 ± 10%) and D-galactose-treated mice (23 ± 16%) (Figure 5B). MaSC differentiation was not assessed in vitro because they cannot form colonies in this assay.

Figure 5. D-galactose alters luminal progenitor cell differentiation in vitro. (A) Representative immunofluorescence images showing distinct colonies formed by luminal progenitor cells on plates pre-seeded with irradiated NIH-3T3 fibroblasts; (B) Bar graph shows the distribution of different types of colonies formed by luminal progenitor from control and D-galactose-treated mice (> 20 colonies per animal × 3 animals). K14: basal cell marker keratin 14; K8: luminal cell marker keratin 8.

D-galactose alters stem/progenitor cell function in vivo and increases hyperplasia in regenerated glands

We further assessed stem cell self-renewal and differentiation by the cleared fat pad (CFP) transplant assay. Transgenic GFP C57BL/6 mice were treated with control or D-galactose, and MaSCs and LPs were subsequently isolated as donors for in vivo transplant. We have previously shown that 3D organoids contain stem cells that can repopulate CFP [23]. Thus, 3D organoids were used for in vivo transplantation. Regenerated glands were clearly visible with the fluorescence microscope given their expression of GFP (Figure 6A). For MaSCs, upon transplant of one single 3D organoid per CFP, we obtained six positive outgrowths from six transplants in the control MaSCs, representing 100% engraftment frequency, but only three positive outgrowths (50% engraftment) in MaSCs derived from the D-galactose-treated mice (Figure 6B). For LPs, we injected five individual 3D organoids per CFP into six CFPs and obtained two positive outgrowths (33% engraftment) in the control LPs, but none in the D-galactose-treated LPs (Figure 6B). The regenerated glands were further harvested for flow cytometry analysis of the luminal-to-basal cell ratio as well as histopathological analysis. Our analysis showed a decreased luminal-to-basal ratio of regenerated glands from D-galactose-treated MaSCs (1.1 ± 0.2) when compared to those from control MaSCs (2.4 ± 0.1) (Figure 6C). Similar to primary mammary glands, regenerated mammary ducts formed by D-galactose-treated MaSCs also showed a higher proportion of hyperplastic ducts than those from control MaSCs (37.5 ± 1.5% vs. 17.6 ± 1.9%) (Figure 6C).

Figure 6. D-galactose alters mammary stem/progenitor cell function in vivo and increases hyperplasia in regenerated glands. (A) Regenerated glands from in vivo transplant of one basal 3D organoid from GFP mice with or without D-galactose treatment (scale bars, 1 mm); (B) Positive take of outgrowth from control or D-galactose-treated stem/progenitor cells in the cleared fat pad (CFP) transplant assay; (C) Luminal-to-basal cell ratio in mammary epithelial cells isolated from regenerated glands derived from control and D-galactose-treated GFP mice (n = 3–4); and (D) H&E histology analysis shows % hyperplasia in mammary ducts from regenerated glands derived from control and D-galactose-treated GFP mice (n = 3–6). Asterisks, significant difference between control and D-galactose (^*P < .05).

Animals

Animal care and use were conducted according to established guidelines approved by the Institutional Animal Care and Use Committee at Wenzhou Medical University (Approval number: 2018-211). Wild type C57BL/6 mice, purchased from Nanjing Medical University, were housed at 20–25°C in a light-controlled (12/12 h) room with unrestricted access to water and food. Transgenic green fluorescent protein (GFP) C57BL/6 mice were originally obtained from Jackson Laboratory and raised in our facility.

D-galactose aging model

Accelerated aging in mice was induced by daily subcutaneous injection of 100 mg/kg of D-galactose for 45 days. The control mice were injected with phosphate buffer saline (pH 7.4). The injection was started at three months old and five mice were used for each treatment group. For the in vivo transplant experiment, GFP C57BL/6 mice were used for control or D-galactose treatment.

Enzyme-linked immunosorbent assay (ELISA)

Blood was collected by venipuncture and allowed to clot at room temperature. Serum was collected after centrifugation at 700× g for 10 min and stored at −80°C before use. The levels of IL-6 and TNF-α were measured using corresponding ELISA kits from Abcam according to user instructions, and the OD value was read at 450 nm using a BioTek spectrophotometer.

Quantification of ductal hyperplasia

Normal ductal structures are characterized by an outside myoepithelial cell layer and an inside luminal epithelial cell layer. Hyperplastic ducts are characterized by more than two layers of epithelial cells (Figure 2B). The percentage of hyperplasia was calculated by dividing the number of ductal structures showing hyperplastic lesions by the total number of ducts counted. A minimum of 50 ductal structures were counted for each gland.

Immunohistochemistry

Mammary glands were fixed for 36 h in 10% neutral-buffered formalin, dehydrated in ethanol, and embedded in paraffin wax. Tissue sections of 4 μm on glass slides were processed as described previously [13]. After antigen retrieval and blocking of nonspecific binding, sections were incubated with recombinant anti-STAT5 (phosphor Y694) antibody [E208] (ab32364, Abcam) (dilution 1:100) overnight at 4°C, and then washed and incubated with biotin conjugated secondary antibodies for 1 h at room temperature. Sections were further incubated with streptavidin-horseradish peroxidase for 30 min and stained with diaminobenzidine for 15 min before dehydration and mounting.

Whole-mount carmine staining

Mammary glands were dissected, spread onto glass slides, and fixed overnight in Carnoy’s fixative at room temperature and processed as previously described [13].

Mammary epithelial cell preparation and flow cytometry analysis

Mammary inguinal and thoracic glands were harvested and processed to generate single cell suspensions for flow cytometry (FACSAria-IIIu, BD Biosciences) analysis as previously described [23, 34]. Mammary stem/progenitor cells were enriched and isolated from endothelial (CD31) and hematopoietic (CD45 and TER119) lineage-depleted (Lin^-) mammary epithelial cells using cell surface markers CD24 and CD49f. Basal stem cells are enriched in basal cells characterized by CD24^lowCD49f^high and luminal stem cells are enriched in luminal cells characterized by CD24^highCD49f^low.

In vitro serial passage of 3D organoids

Sorted basal and luminal cells were first used for mammary sphere culture and subsequently followed by sphere differentiation in Matrigel 3D culture [23]. To assess stem cell self-renewal function in vitro, 3D organoids formed in Matrigel culture (P0) were used for in vitro serial passage as we described previously [35]. The serial passage process was repeated for five generations (P5), and the number of colonies that can be continuously passaged to P5 was tabulated against the total number of colonies assessed. For each type of 3D organoids (basal or luminal), 10 organoids per animal × 3 animals, a total of 30 organoids were assayed.

Mammary colony forming cell assay

The mammary colony forming cell assay is used to assess the differentiation function of luminal progenitor cells. It is known that luminal progenitor cells can form distinct colonies when cultured on plates pre-seeded with irradiated NIT-3T3 fibroblasts [23, 35]. In this study, the colony forming cell assay was performed [13]. However, instead of Giemsa staining, colonies were stained with basal and luminal markers of keratin 14 (K14) and K8, respectively, using immunofluorescence (IF). In detail, colonies were fixed with 100% cold methanol for 1 min, washed with phosphate buffer solution with 0.05% Tween 20 (PBST), blocked with 10% serum/1%BSA/0.3M glycine in 0.1% PBS-Tween 20 for 30 min, and incubated with primary antibodies of K14 (rabbit monoclonal IgG, ab119695, 1:200 dilution, Abcam) and K8 (rabbit monoclonal IgG, ab53280, 1:200 dilution, Abcam) diluted in 5% serum/1%BSA/0.3M glycine in 0.1% PBS-Tween 20 at 4°C overnight. Colonies were then washed with PBST twice before being incubated with secondary antibodies diluted in 1%BSA/0.3M glycine in 0.1% PBS-Tween 20 at room temperature for 1 h. Finally, colonies were washed with PBST and stained with DAPI for fluorescent imaging. A minimum of 20 colonies per animal (× 3 animals) per treatment group were assessed with IF.

Cleared fat pad transplant

Stem cell self-renewal and differentiation in vivo was assessed by the cleared fat pad transplant assay as described previously [13]. In brief, 3D organoids from control and D-galactose-treated GFP C57BL/6 mice were injected into the contralateral inguinal glands of 21-day-old virgin wild-type female C57BL/6 mice cleared of endogenous epithelium. For basal stem cells, one single solid 3D organoid was injected into one cleared fat pad (CFP). For luminal progenitor cells, five hollow 3D organoids were injected into one CFP. A total of six CFPs were used for each type of 3D organoid per treatment group. Outgrowths, defined as epithelial structures with both ductal and lobular structures, were evaluated by Nikon fluorescence microscope after eight to ten weeks. The regenerated glands were further harvested for flow cytometry analysis of the luminal-to-basal cell ratio as well as histopathological analysis for hyperplastic lesions.

Statistical analysis

Data were analyzed using GraphPad Prism 6.0 (GraphPad Software Inc., La Jolla, CA). Differences between various treatment groups were evaluated with a non-paired t-test. Results are presented as mean ± SD. Statistical significance was defined by P < .05.