Fig 2: An optimized workflow to efficiently purify and characterize SCs and fibroblasts from adult human peripheral nerve tissue. This protocol uses heterogeneous human cultures containing cells identifiable as SCs and fibroblasts as evidenced by expression of p75NGFR, a SC (neural crest)-specific receptor. MACS of p75-labelled cells allowed the fast recovery of SCs and fibroblasts retaining their viability and biological activity. The rounds of MACS required to achieve > 95% SC purity was dependent on the degree of fibroblast growth (high or low, as indicated). Immunofluorescence microscopy, flow cytometry and transcriptome (RNA-seq) analysis were used to confirm the identity and purity of the MACS-purified cells. Some key lineage- and stage-specific genes revealed by RNAseq (bottom panel) emphasized the independent origin of human SCs and fibroblasts, their relative immaturity, and the heterogeneity of the fibroblast populations. The genes highlighted in color were confirmed by immunological methods.

Fig 3: Purification of human SC cultures via MACS. Results from representative experiments using unpurified human SC populations containing high (a) and low (b–c) fibroblast contamination are shown. Cells were labeled in suspension with p75NGFR antibodies and separated using MS-columns (Methods). Samples from the pre-sorting (total) and post-sorting cells were plated in SC growth medium (retained cells) and FBS-only medium (eluted cells) for analysis by immunofluorescence microscopy and flow cytometry. The retained and eluted cells were highly viable (c), rapidly adhered to the substrate acquiring the expected pattern of growth (a–c) and underwent proliferation, as denoted by EdU labeling (red nuclei, c). Representative images of a senescent human SC culture (passage-4) subjected to MACS is shown in panel c (bottom images, SA-ßGAL activity) to confirm the absence of senescent cells in the eluted fraction. Observe the negligible representation of S100ß and p75NGFR positive cells in the populations of eluted cells (a–c) and the high SC enrichment obtained in one round of purification (a). A quantification of the proportion of proliferating (EdU positive) cells discriminated on the basis of p75NGFR expression (green) is shown in panel (d) along with an assessment of viability (Trypan blue exclusion assays) for cells in suspension prior to (total) and immediately after MACS (n = 8).

Fig 4: HER2 CAR-NK cells have greater killing against HER2-positive breast cancer target cells compared to HER2 CAR-T cells(A) Representative histograms of HER2 CAR and NGFR surface expression in donor-matched NK cells and T cells.(B) Transgene expression of HER2 CAR and NGFR on NK cells, CD4+ and CD8+ T cells.(C) Cell-mediated cytotoxicity against SKBR3 cells was measured and relative change in specific lysis for HER2 CAR- and control vector transduced-NK cells was compared to HER2 CAR-T cells.(D and E) IFN-γ (D) and TNF-α (E) release by HER2 CAR-, control vector transduced-, and non-transduced NK cells or T cells in response to SKBR3 target cells. Data represent mean ± SEM from two independent experiments with four donors per condition.∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (B, two-way ANOVA with Sidak's post hoc tests; C, one-way ANOVA with Dunnet's post hoc tests; D and E, two-way ANOVA with Tukey's post hoc tests). ns, no significant difference.

Fig 5: HR cultured mammary epithelial cells show luminal expansion with a basal phenotype.a–d, Flow cytometry analysis of passage 4 epithelia stained for CD271 and MUC1 from an AR 19-year-old woman and an AR 66-year-old woman (a), HR women of different ages (31, 35, 40 and 53 years) harboring BRCA1 mutations (b), an HR 52-year-old woman harboring a PALB2 mutation (PALB2mut) (c) and an HR 33-year-old woman harboring a BRCA2mut (d). e, Dot plot of the percentage of LEp populations from passage 4 epithelial strains derived from AR younger women (=35 years, n = 17), AR middle-aged women (36–55 years, n = 9), AR older women (>55 years, n = 10) and HR women (n = 15). The median of each sample is indicated by a horizontal line. The P values were computed using the Kruskal–Wallis test adjusted for multiple comparisons with Dunn’s post-hoc test. f–j, Immunofluorescence staining of passage 4 cultured epithelial cells stained for KRT19 (green), KRT14 (red) and Hoechst (blue) from an AR 19-year-old woman (f), an AR 66-year-old woman (g), an HR 24-year-old woman harboring a BRCA1mut (h), an HR 33-year-old woman harboring a BRCA2mut (i) and a 52-year-old woman harboring a PALB2mut (j). The image on the right-hand side of each panel is a magnification of the area outlined by the white rectangle in the merged image. Scale bars, 50 µm. Experiments in f–j were repeated three times independently with similar results.