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. 2009 Jul 9;114(2):279-89.
doi: 10.1182/blood-2009-02-203638. Epub 2009 May 11.

Zebrafish kidney stromal cell lines support multilineage hematopoiesis

David L Stachura  1 , Jason R Reyes, Petr Bartunek, Barry H Paw, Leonard I Zon, David Traver
Affiliations

Zebrafish kidney stromal cell lines support multilineage hematopoiesis

David L Stachura et al. Blood. .

Abstract

Studies of zebrafish hematopoiesis have been largely performed using mutagenesis approaches and retrospective analyses based upon gene expression patterns in whole embryos. We previously developed transplantation assays to test the repopulation potentials of candidate hematopoietic progenitor cells. We have been impaired, however, in determining cellular differentiation potentials by a lack of short-term functional assays. To enable more precise analyses of hematopoietic progenitor cells, we have created zebrafish kidney stromal (ZKS) cell lines. Culture of adult whole kidney marrow with ZKS cells results in the maintenance and expansion of hematopoietic precursor cells. Hematopoietic growth is dependent upon ZKS cells, and we show that ZKS cells express many growth factors and ligands previously demonstrated to be important in maintaining mammalian hematopoietic cells. In the absence of exogenous growth factors, ZKS cells maintain early hematopoietic precursors and support differentiation of lymphoid and myeloid cells. With the addition of zebrafish erythropoietin, ZKS cells also support the differentiation of erythroid precursors. These conditions have enabled the ability to ascertain more precisely the points at which hematopoietic mutants are defective. The development of robust in vitro assays now provide the means to track defined, functional outcomes for prospectively isolated blood cell subsets in the zebrafish.

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Figures

Figure 1
Figure 1
ZKS cells are primary kidney stromal cells. (A) Morphologic characterization of ZKS cells. (i,ii) May-Grünwald/Giemsa staining of ZKS cells on glass coverslips reveals stromal morphology. Images in subpanel i were photographed at 100× at 100% confluence; those in subpanel ii were photographed at 400× at low confluence. Transmission electron micrographs of ZKS stromal cells show ultrastructural features of fibroblasts. (iii) Close-up of ZKS cell, 7900×. Note abundant mitochondria and free ribosomes. (iv) Close-up of a ZKS cell, 19 000×. Note abundant Golgi and rough endoplasmic reticular network. n indicates nucleus; g, Golgi; r, ribosome; rER, rough endoplasmic reticulum; ly, lysosome; f, filaments; and m, mitochondria. (B) ZKS cells do not express hematopoietic specific transcripts. Gene expression analysis of ZKS cells by RT-PCR for the pan leukocytic transcript cd45 and erythroid specific gata1. Gene names listed at left. Whole kidney used as positive controls, dH2O as negative controls. (C) Gene expression analysis of ZKS cells by RT-PCR for transcripts involved in proliferation and differentiation of progenitor cells (left column), niche signaling and lymphoid development (middle column), and lineage-specific signaling, maintenance, and differentiation (right column). Gene names listed at left. Whole kidney used as positive controls, dH2O as negative controls.
Figure 2
Figure 2
ZKS cells support hematopoiesis. (A) All hematopoietic cell populations are represented in CFSE+ cultures. Representative flow cytometric light scatter profile showing the different populations of cells present in CFSE-labeled WKM at day 0, before plating on ZKS. (B) ZKS support cell survival. Total cell numbers of WKM cultured with ZKS cells (—▲—) and without (---●---) over time (n = 4). (C) Lymphoid, precursor, and myeloid cells all survive over time. Total number of lymphoid (left), precursor (center), and myeloid (right) cells present in WKM cultures with ZKS cells (—▲—) and without (---●---; n = 4). In all cases, total cell numbers are given at left, and days in culture are given at bottom. In all cases, the graph depicts the mean and SD for 4 independent replicates.
Figure 3
Figure 3
ZKS cells support hematopoiesis; morphology and gene expression data. (A) Representative hematopoietic cell types recovered from in vitro culture on ZKS stroma. May-Grünwald/Giemsa stained cells were photographed at 1000×. After photographing, cells were cut and pasted from multiple fields to create composite image. (B) Gene expression analysis of cells by RT-PCR recovered from ZKS cultures for the panleukocytic transcript cd45, the erythroid-specific transcript gata1, myeloid-specific transcripts pu.1 and mpx, and the lymphoid-specific transcripts pax5, igM, gata3, lck, and rag2. Gene names listed at left, days in culture on ZKS stroma at top. Whole kidney used as positive controls, dH2O as negative controls. (C) Percentages of lineages in recovered cell populations over 11 days of culture. Percent population listed at left, days in culture on ZKS stroma at bottom. Green indicates myeloid; purple, precursor; blue, lymphoid; and red, erythroid.
Figure 4
Figure 4
ZKS cells stimulate differentiation of hematopoietic precursors. (A) Populations containing hematopoietic progenitors differentiate in culture on ZKS cells. Lymphoid fraction from βactin:GFP+ WKM, which contains rare HSCs, was sorted, plated, and analyzed after culture for the time period indicated at top (top row). βactin:GFP+ WKM precursor fraction, which contains erythroid, myeloid, and lymphoid progenitors was sorted, plated, and analyzed after culture for time period indicated at top (bottom row). Reanalysis of sorted GFP+ lymphoid and precursor fractions from βactin:GFP+ WKM (day 0) shows 99% purity. Data are representative of 3 independent experiments. Scatter gates drawn as in Figure 2. (B) Total number of GFP+ lymphoid (left), precursor (center), and myeloid (right) cells present over time from cultured βactin:GFP+ lymphoid (top, circles) and precursor (bottom, diamonds) fractions (n = 3). Each graph depicts the mean and SD for 3 independent replicates.
Figure 5
Figure 5
ZKS cells stimulate differentiation of zebrafish hematopoietic precursors; morphologic evidence. (A) Representative hematopoietic cells recovered from purified βactin:GFP+ lymphoid (top) and precursor (bottom) fractions cultured on ZKS cells stained with May-Grünwald/Giemsa. All images were photographed at 1000×. After photographing, cells were cut and pasted from multiple fields to create composite image. (B) Percentages of lineages derived from lymphoid fraction (left) and precursor fraction (right) recovered cell populations over 8 days of culture. Percent population listed at left, days in culture on ZKS stroma at bottom. Blue indicates lymphoid; purple, precursor; and green, myeloid.
Figure 6
Figure 6
zEpo increases precursor cell survival and differentiation. (A) Epo stimulates precursor survival. Fold change in precursor number after 5 days in culture. Cytokine conditions listed at bottom (n = 3). (B) RT-PCR expression of the zebrafish transferrin-a (tfa) gene in zebrafish tissues. (C) Increased erythroid differentiation achieved by exogenous iron delivery. Percent o-dianisidine-positive erythroid cells after 5 days of culture with erythroid stimulating factors. Factors added to media listed at bottom (n = 3). In all cases, the graph depicts the mean and SD for 3 independent replicates.
Figure 7
Figure 7
Fe-SIH rescues erythroid differentiation in cdyte216/te216 mutant WKM cultured on ZKS cells. (A) Morphologic (May-Grünwald/Giemsa, top row) and histochemical (o-dianisidine, bottom row) staining of cdy te216/216 mutant WKM cultured on ZKS for 5 days with no factors added (left column) or 100 μg zEpo and Fe-SIH added (right column). Arrowheads denote erythroid cells. (B) Fold expansion of precursor cells from erythroid mutants over 5 days in culture on ZKS cell without (−) or with (+) recombinant Epo and Fe-SIH (n = 4). (C) Percent o-dianisidine-positive erythroid cells after 5 days of culture without (−) or with (+) recombinant Epo and Fe-SIH (n = 4; *P < .01). In all cases, the graph depicts the mean and SD for 4 independent replicates.
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