PrP is expressed on bone marrow populations enriched in HSC activity. (A) Freshly isolated BM cells were stained with Hoechst dye 33342, and the SP fraction was gated (gates 1 and 2) to analyze the expression of PrP. In plot 1, forward scatter (FSC) and side scatter (SSC) is used to gate on hematopoietic cells. Hoechst Red and Hoechst Blue (plots 2 and 2a, gate 2 was set as 0.02% of total cells) represent two fluorescence emission wavelengths used to detect the SP cells. Plot 2a is an expansion of the gate 2 region of plot 2. PrP-null BM SP cells served as a negative control for PrP antibody staining (plot 3). WT BM SP cells were stained for PrP (plots 4–12) together with isotype control (plot 4), Endoglin (plot 5), Sca-1 (plot 6), CD43 (plot 7), CD44 (plot 8), CD49D (plot 9), CD49E (plot 10), CD11A (plot 11), or CD62L (plot 12). (B) Total BM cells were stained with anti-Endoglin followed sequentially by anti-rat-PE/CY5.5, a mixture of biotinylated lineage-specific antibodies, and streptavidin–APC, anti-PrP-FITC, and anti-Sca-1-PE. Plot 1 shows the gate of FSC and SSC channels. The lowest 5% of APC-stained cells (i.e., Lin⁻) were gated (plot 2). Plot 3 shows the staining of the gated Lin⁻ cells with Sca-1 and Endoglin, and plot 4 shows the staining of the gated Lin⁻ Sca-1⁺ Endoglin⁺ cells with PrP. Plot 5 shows the staining of the gated Lin⁻ Sca-1⁺ Endoglin⁻ cells with PrP.

All LT repopulating bone marrow HSC cells express surface PrP. A total of 1 × 10⁵ PrP⁻ or 2 × 10⁴ PrP⁺ CD45.2 donor BM cells (A), 2 × 10⁴ PrP⁺ CD45.2 donor BM cells (B), 500 sorted Lin⁻Sca-1⁺PrP⁻ or 100 Lin⁻Sca-1⁺PrP⁺ CD45.2 donor BM cells (C), or 500 isolated SP PrP⁻ or 250 SP PrP⁺ donor BM cells (D) were mixed with 1 × 10⁵ competitor CD45.1 cells and transplanted into lethally irradiated CD45.1 mice (n = 4–5). (A) Donor CD45.2 contribution at 4 weeks and 6 months after transplant. (B) Multilineage contribution at 6 months after transplant. (C) Donor contribution at 3 weeks and 4 months after transplant. (D) Donor contribution at 4 months after transplant.

Ectopic expression of PrP rescues the impaired self-renewal of PrP-null stem cells during serial transplantation. (A) Colony assays for erythroid, lymphoid, and myeloid progenitors in PrP-null and WT BM. Total BM cell populations were plated in methylcellulose medium M3434 (StemCell Technologies) for quantifying CFU-GEMM, CFU-GM, and BFU-E colonies, and in M3630 (StemCell Technologies) for quantifying CFU-Pre-B colonies. (B) Serial transplantation of PrP-null and WT BM with competitor bone marrow cells. A total of 2 × 10⁶ donor CD45.2 PrP-null or littermate control BM cells were mixed with 2 × 10⁶ CD45.1 WT BM cells and transplanted into lethally irradiated CD45.1 recipients (n = 6). The extent of chimerism in peripheral blood (bars 1 and 2) was analyzed 4 months after transplant. In the experiment in bars 3 and 4, BM cells from the primary transplanted mice were pooled, and 2 × 10⁶ cells were injected directly into each of five lethally irradiated CD45.1 recipients. The fraction of donor CD45.2 cells in the peripheral blood of these transplanted mice was analyzed 4 months later. The process was repeated for the tertiary transplants (bars 5 and 6). This is a combined result of three independent experiments from a total of initial six null or wild-type control mice. ∗, Significantly different from bar 3 value, P < 0.005; ∗∗, significantly different from bar 5 value, P < 0.005. (C) Serial transplantation of PrP-null and WT BM cells without competitors; rescue of HSC activity in PrP-null cells by PrP expression. A total of 1 × 10⁶ PrP-null or WT BM CD45.2 cells, pooled from three donors, were transplanted into lethally irradiated CD45.1 recipients without competitors. Recipients were monitored daily for survival for >30 days (bars 1 and 2, n = 6). These mice were killed after 4 months. From them, 5 × 10⁵ BM cells were collected and transplanted into new irradiated recipients (bars 3 and 4, n = 7). The process was repeated an additional time for tertiary transplants (bars 5 and 6, n = 12). In parallel, 1 × 10⁶ PrP-null BM cells isolated from the surviving secondary transplant recipients, as shown in bar 3, were infected by retroviruses encoding GFP, PrP, or PrP Δ23-72, and injected into irradiated recipients (bars 7–9, n = 7–8). Plotted is the fraction of surviving mice 50 days after each bone marrow transplant. See Fig. 6 for details of animal survival. (D) A total of 1 × 10⁶ BM cells from the secondary transplanted mice shown in bars 3 and 4 of C were transplanted into the lethally irradiated recipients. Survival data were plotted as Kaplan–Meier curves (n = 11 for each group, P < 0.0001, log-rank test). (E) Competitive transplantation demonstrates impaired renewal of PrP-null HSC activity during successive bone marrow transplants. Here, 5 × 10⁵ PrP-null or WT BM collected from primary transplanted mice 4 months after transplant (without competitors, as in C, bars 1 and 2) were mixed with 5 × 10⁵ CD45.1 freshly isolated BM cells and transplanted into lethally irradiated recipients. Peripheral blood engraftment at 6 weeks and 5 months after transplant is shown (n = 4). ∗, Significantly different from bar 1 value, P < 0.005; ∗∗, significantly different from bar 3 value, P < 0.05.

PrP-null HSCs are more sensitive than normal to myelotoxic injury. (A) A total of 5 × 10⁵ CD45.2 PrP-null or wild-type BM cells, pooled from three littermates, were transplanted into lethally irradiated CD45.1 recipients. One month after transplant, 11 recipient mice from each group were treated with 150 mg/kg 5-FU i.p. weekly for 2 weeks. The survival of these two groups was analyzed by using a log-rank nonparametric test (P = 0.0307, n = 11 in each group) and expressed as Kaplan–Meier survival plot. (B) 5-FU (150 mg/kg) was administered i.p. to three wild-type CD45.2 mice. After 3 days, the BM of these treated mice was pooled and fractionated according to PrP expression. A total of 1 × 10⁵ PrP⁺ or 1 × 10⁶ PrP^× BM cells were transplanted, together with 2 × 10⁵ competitor CD45.1 wild-type BM cells, into lethally irradiated CD45.1 recipients. The level of chimerism in the peripheral blood of the recipients was analyzed 4 months later (n = 5).