Hematopoietic activity of a stromal cell transmembrane protein containing epidermal growth factor-like repeat motifs - PubMed (original) (raw)

Hematopoietic activity of a stromal cell transmembrane protein containing epidermal growth factor-like repeat motifs

K A Moore et al. Proc Natl Acad Sci U S A. 1997.

Abstract

Primitive hematopoietic stem cells are closely associated with discrete in vivo microenvironments. These "niches" are thought to provide the molecular signals that mediate stem cell differentiation and self-renewal. We have dissected the fetal liver microenvironment into distinct cellular components by establishing an extensive panel of stromal cell lines. One particular cell line maintains repopulating stem cells for prolonged in vitro culture periods. A subtraction cloning strategy has yielded a cDNA that encodes a cell surface glycoprotein with a restricted pattern of expression among stromal cell lines. This molecule, previously identified as delta-like/preadipocyte factor-1, contains epidermal growth factor-like repeats that are related to those in the notch/delta/serrate family of proteins. We have investigated the potential role of this molecule in hematopoietic stem/progenitor cell regulation. We show that the delta-like protein displays activity on purified stem cells by promoting the formation of "cobblestone areas" of proliferation. These cobblestone areas contain both primitive high-proliferative potential progenitors and in vivo repopulating stem cells.

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Figures

Figure 1

Dlk expression analysis in stromal cell lines. (A) (Upper) A 1.6-kb dlk transcript is visualized in the parental AFT024 and 2012 cell lines and their subclones, but not in 2018, CFC034 and BFC012. (Lower) The same filter hybridized with a β-actin probe. (B) RT-PCR analysis of 14 fetal liver-derived stromal cell lines and other cell lines.

Figure 2

CSA formation by hematopoietic stem cells in the presence of soluble dlk protein. Data are from four experiments: two each with adult BM cells (Sca-1+, c-kit+, linlo/−), and day 14 fetal liver cells (AA4.1+, Sca-1+, c-kit+, linlo/−). Results are expressed as the ratio/fold-increase in CSA number for 14 data points each for the four different experiments. Error bars represent SEM. ∗, P = 0.01 comparing dlk vs. none to none vs. control; ∗∗, P = 0.001 comparing dlk vs. control to none vs. control (Student’s t test).

Figure 3

Membrane-bound dlk expression in transfected BFC012 cells. (Left) A flow diagram of dlk expression in transfected BFC012 populations (BFC-dlk) and cells transfected with the selection plasmid alone (BFC-Zeo). (Right) Expression of dlk in a cloned line (BFC-dlk-5) derived from the expressing population and a control clone (BFC-Zeo-1).

Figure 4

CSA formation by hematopoietic stem cells in the presence of membrane-bound dlk. (A) Bars labeled BFC are from five groups (nontransfected BFC012 cells, two control pZeo-transfected BFC012 populations, and two clones derived from the pZeo-transfected populations). Bars labeled BFC-dlk are from three groups shown to express transfected dlk; one dlk-transfected BFC012 population and two individual transfected clones. Error bars represent SEM. ∗∗, P < 0.001 days 3, 4, and 5; ∗, P < 0.01 days 6 and 7 (Student’s t test). (B) A clone derived from the dlk-transfected populations of BFC012 cells (BFC-dlk-5) and a clone derived from pZeo-transfected populations (BFC-Zeo-1) were used for CSA assay with purified fetal liver stem cells. CSA/1000 input stem cells are expressed as the mean of three individual experiments, error bars represent the SEM. ∗∗, P < 0.001 at days 4, 6, and 8 (Student’s t test).

Figure 5

HPP multilineage clonogenic progenitors and in vivo repopulating stem cells are maintained in short-term dlk-expressing cocultures. (A) Fetal liver stem cells were purified as described and assayed for their progenitor content immediately after purification and after culture on BFC-dlk-5 and BFC-Zeo-1. At day 4, the cultures were used for clonogenic progenitor (three experiments) and transplantation assay (two experiments). Bars represent data from three experiments with day 0 cells (Fresh) and day 4 cocultured cells (BFC-dlk-5 and BFC-Zeo-1), error bars represent SEM. ∗, P = 0.01 for total CFU-C from fresh stem cells compared with total CFU-C from BFC-dlk-5 cocultures at day 4; ∗∗, P = 0.001 for total CFU-C from BFC-dlk-5 compared with total CFU-C from BFC-Zeo-1 (Student’s t test). (B) Analysis of in vivo repopulating ability of purified fetal liver stem cells cocultured for 4 days on BFC-dlk-5, BFC-Zeo-1, and AFT024 monolayers. Results are from nine individual mice in two experiments (4–5 mice in each experiment) at 10 weeks after transplantation. P = 0.05 for BFC-dlk-5 vs. BFC-Zeo-1 (Student’s t test).

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Hematopoietic activity of a stromal cell transmembrane protein containing epidermal growth factor-like repeat motifs - PubMed (original) (raw)