Identification of fetal liver stroma in spectral cytometry using the parameter autofluorescence

Abstract

The fetal liver (FL) is the main hematopoietic organ during embryonic development. The FL is also the unique anatomical site where hematopoietic stem cells expand before colonizing the bone marrow, where they ensure life-long blood cell production and become mostly resting. The identification of the different cell types that comprise the hematopoietic stroma in the FL is essential to understand the signals required for the expansion and differentiation of the hematopoietic stem cells. We used a panel of monoclonal antibodies to identify FL stromal cells in a 5-laser equipped spectral flow cytometry (FCM) analyzer. The "Autofluorescence Finder" of SONY ID7000 software identified two distinct autofluorescence emission spectra. Using autofluorescence as a fluorescence parameter we could assign the two autofluorescent signals to three distinct cell types and identified surface markers that characterize these populations. We found that one autofluorescent population corresponds to hepatoblast-like cells and cholangiocytes whereas the other expresses mesenchymal transcripts and was identified as stellate cells. Importantly, after birth, autofluorescence becomes the unique identifying property of hepatoblast-like cells because mature cholangiocytes are no longer autofluorescent. These results show that autofluorescence used as a parameter in spectral FCM is a useful tool to identify new cell subsets that are difficult to analyze in conventional FCM.

Keywords: autofluorescence; fetal liver; hepatoblast-like cells/hepatocytes; spectral flow cytometry.

FIGURE 1

Gating strategy to identify stellate cells, hepatoblasts‐like cells, hepatocytes and cholangiocytes. E18.5 mouse fetal liver cell suspensions were stained with biotin labeled antibodies recognizing CD45, TER119, CD71, CD117. Negative cells were magnetically sorted and labeled with the antibodies in Table 1. (A) The spectra of emission of fetal liver cells after depletion (marked with streptavidin in a dump channel) and gated out of PI labeled cells. (B) After unmixing, cells were manually gated to define endothelial, hepatic and stellate cells. The analysis started with a FSC_A/SSC_A gate, doublet exclusion and PI positive/SAV positive cell exclusion. The gate for SAV exclusion was set by the depletion control sample (not depleted). CD324 was defined as a marker of epithelial cells, cholangiocytes CD324^highCD326⁺; hepatocytes CD324^lowCD326⁻; hepatoblasts‐like cells CD324^highCD326⁻. Stellate cells are defined by as PDGFRa⁺ CD166⁺. [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 2

Autofluorescence finder. The analytical tool autofluorescence applied to unstained cells before unmixing. (A) Autofluorescence is defined manually in cells that show fluorescence in the different lasers, as defined in Materials and Methods. (B) The spectra of emission of the two defined autofluorescent (AF) signals in the different lasers. [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 3

Two distinct autofluorescent signals: AF‐1 defines stellate cells and AF‐2 defines a subset of hepatic cells and cholangiocytes. After unmixing the two AF signals can be used as two additional fluorescent parameters. (A) E18.5 FL live CD45⁻TER119⁻CD71⁻CD117⁻ cells were analyzed as in Figure 1B. (B) Analysis of E18.5 FL live CD45⁻TER119⁻CD71⁻CD117⁻AF1⁺ cells using the same strategy as in Figure 1B. (C) Analysis of E18.5 FL live CD45⁻TER119⁻CD71⁻CD117⁻AF2⁺ cells using the same strategy as in Figure 1B. (D) Analysis of E18.5 FL live CD45⁻TER119⁻CD71⁻CD117⁻AF1⁻AF2⁻ cells using the same strategy as in Figure 1B. [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 4

Autofluorescence is a cell intrinsic property. (A) Fetal liver cells negative for the dump channel and for PI expressing cells were sorted after being stained with a simplified antibody panel including Gp38, CD140a, NG2, CD166, CD324 and CD326. Hepatocytes, hepatoblast‐like cells and stellate cells were sorted according to the strategy shown (presort). Sorted cells were reanalyzed in the ID7000 spectral analyzer (sorted populations). Left plots show purity control, right plots show AF of sorted cells. (B) Sorted cholangiocytes, hepatoblast‐like cells and stellate cells as in A were subjected quantitative RT‐PCR in triplicate samples of two independent experiments for Sox9, Onecut1, Alb, Afp, Desmin and Pdgfrb, and normalized to the expression of β‐actin used as house‐keeping gene. Data are represented as mean +/− SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 5

AF2 defines hepatoblast‐like cells after birth. Analysis of live CD45⁻TER119⁻CD71⁻CD117⁻ P3 and P9 liver cells in a plot of CD324/CD326 after the conventional gating strategy defined in Figure 1 (left plots) or on the cells gated for AF2 expression (right plots). [Color figure can be viewed at wileyonlinelibrary.com]