SACK-expanded hair follicle stem cells display asymmetric nuclear Lgr5 expression with non-random sister chromatid segregation

Abstract

We investigated the properties of clonally-expanded mouse hair follicle stem cells (HF-SCs) in culture. The expansion method, suppression of asymmetric cell kinetics (SACK), is non-toxic and reversible, allowing evaluation of the cells' asymmetric production of differentiating progeny cells. A tight association was discovered between non-random sister chromatid segregation, a unique property of distributed stem cells (DSCs), like HF-SCs, and a recently described biomarker, Lgr5. We found that nuclear Lgr5 expression was limited to the HF-SC sister of asymmetric self-renewal divisions that retained non-randomly co-segregated chromosomes, which contain the oldest cellular DNA strands, called immortal DNA strands. This pattern-specific Lgr5 association poses a potential highly specific new biomarker for delineation of DSCs. The expanded HF-SCs also maintained the ability to make differentiated hair follicle cells spontaneously, as well as under conditions that induced cell differentiation. In future human cell studies, this capability would improve skin grafts and hair replacement therapies.

Figure 1. Examples of complete data profiles for a cytochalasin D (CD) assay of 3C5 and 5B8 clonal hair follicle stem cell strains cultured under conditions that either promote or suppress asymmetric self-renewal.

Co-ISIF results with anti-cyclin A (CyA, red) and anti-LGR5 (green) antibodies are indicated by a square arrangement of four circles. Horizontally, the paired circles represent paired sister nuclei in a CD-arrested binucleated cell. The top pair indicates the fluorescence pattern observed for cyclin A; and the bottom pair indicates the corresponding staining pattern for LGR5. Open circles indicate that no significant fluorescence was detected. Closed circles indicate that fluorescence was detected for the respective biomarker. All 10 theoretically possible staining patterns are depicted (a–j). (A) conditions that promote asymmetric self-renewal (-Xn); (B) conditions that suppress asymmetric self-renewal (+Xn). n, total number of binucleated cells evaluated and the respective numbers for each possible pattern type; %, percent that each pattern type represents out of the total number of sister nuclear pairs evaluated. The solid rectangle highlights the “Co-SYM” designation for Lgr5 and cyclin A; and the dashed rectangle highlights to the “Co-ASYM” designation.

Figure 2. Nuclear Lgr5 expression is limited to the cycling stem cell sister of hair follicle stem cell asymmetric self-renewal divisions.

Shown are epifluorescence micrographs from parallel sister pair (SPr) and cytochalasin D (CD) analyses for hair follicle stem cell strain 3C5 cultured under conditions that either suppress asymmetric self-renewal divisions (left) or promote them (right). Examples of respective Co-SYM and Co-ASYM patterns of nuclear Lgr5 and cyclin A detection are given. DAPI, nuclear DNA fluorescence. CyA, ISIF with specific antibodies for cyclin A. Lgr5, ISIF with antibodies for Lgr5. ISIF was performed simultaneously for cyclin A and Lgr5. Merge, overlay of DAPI, cyclin A and Lgr5 epifluorescence images. Phase, corresponding phase micrographs. Scale bar, 50 microns.

Figure 3. Label retention-CD analysis for detection of non-random segregation of immortal DNA strands in hair follicle stem cells.

(A) Diagrammed are the three BrdU-labeling schemes (I–III) employed, as described in the text, to evaluate the Xn-dependency of non-random sister chromatid segregation by 3C5 hair follicle stem cells. The number of generation times (GT; 1GT∼24 hours) for each stage of the analyses (horizontal solid arrows and lines) is indicated (dotted arrows). The times of replacement of cultures with BrdU-, thymidine chase (Thd)-, or CD-supplemented culture medium are noted by vertical lines. At the indicated stages, cells were cultured in medium that either promoted asymmetric self-renewal (-Xn [↑ASR]) or suppressed it (+Xn [↓ASR]). (B) Examples of epifluorescence micrographs of cells evaluated in scheme III for the segregation pattern of BrdU-labeled DNA by ISIF (BrdU). Shown are three examples of binucleated cells showing retention and non-random segregation of BrdU-labeled immortal DNA (NRandom) and one example showing symmetric inheritance indicative of random segregation (Random). DAPI, nuclear DNA fluorescence. Merge, overlaid DAPI and anti-BrdU images. Binucleation was confirmed by phase microscopy (Phase). Scale bars, 25 microns. (C) Quantitative summary of the percentage of CD-induced binucleated cells showing non-random segregation of retained BrdU-labeled DNA (%NR). The mean of three independent experiments is plotted for schemes I and II. Error bars denote the standard deviation of the data.

Figure 4. Quantitative evaluation of the suppression of hair follicle stem cells' non-random segregation by the SACK agent Xn.

The pulse-chase label retention-CD experiment described in the text and Fig. 3 was quantified by digital imaging of fluorescence due to anti-BrdU antibodies. (A) Distribution of the mean fluorescence (Fl) intensity of nuclei in mononucleated cells after 24 hours of BrdU-labeling. (B) Distribution of the mean fluorescence intensities of nuclei in binucleated cells chased in Xn-supplemented medium. (C) Distribution of the mean fluorescence intensities of nuclei in binucleated cells chased in Xn-free medium. p, for statistical confidence by the K-S test that the B and C distributions are distinct. (D) Distribution of the % differences between sister nuclei in binucleated cells chased in Xn-supplemented medium. (E) Distribution of the % differences between sister nuclei in binucleated cells chased in Xn-free medium. p, for statistical confidence by the K-S test that the D and E distributions are distinct. M, median. The mean fluorescence intensity for nuclei in binucleated cells that were unlabeled, but otherwise went through the entire ISIF procedure, was 0.7 ± 1.0.

Figure 5. Induced differentiation properties of SACK-expanded, clonal hair follicle stem cell strains.

Strain 3C5 (A) and 5B8 (B) cells were evaluated for expression of Lgr5 and the hair follicle-epidermis biomarkers keratin 5 (K5), keratin 10 (K10), and Filaggrin, either under conditions of active cell proliferation (UNDIFF) or after differentiation by serum reduction (DIFF) as detailed in Methods. Phase, phase micrographs of cells under the two conditions. Shown are immunofluorescence micrographs developed with either single or dual antibodies directed against the indicated specific biomarkers. The colors of a micrograph's biomarker labels match the corresponding fluorescence colors that indicate detection of the respective biomarker in the micrograph. DAPI, all cells were counterstained to detect nuclear DNA by fluorescence for overlay with respective fluorescence micrographs. In LGR5/DAPI micrographs, arrows denote examples of cells with nuclear Lgr5 fluorescence. In dual biomarker micrographs, arrows denote examples of cells that display fluorescence corresponding to both biomarkers. Scale bar, 200 microns.

Figure 6. Spontaneous differentiation properties of SACK-expanded hair follicle stem cells.

(A–C) Phase micrographs of crystal violet-stained colonies grown under SACK agent-free conditions. (A) At colony edges, where there is active cell division, few differentiated cells are present (40X magnification). (B) In the center of colonies, where cells are growth-arrested, a high degree of differentiation is evident by dark purple cells, which contain light-reflective vesicles (40X). (C) Higher magnification micrograph of cells at the periphery of a differentiating colony (100X). (D) Phase micrograph of unstained light reflective vesicles (arrows; 100X). (E) Oil red-positive vesicles (arrows; 100X). (F) Transmission electron micrograph showing example of a primary cilium (arrow).