Hepatic thrombopoietin is required for bone marrow hematopoietic stem cell maintenance

Abstract

Hematopoietic stem cell (HSC) maintenance depends on extrinsic cues. Currently, only local signals arising from the bone marrow niche have been shown to maintain HSCs. However, it is not known whether systemic factors also sustain HSCs. We assessed the physiological source of thrombopoietin (TPO), a key cytokine required for maintaining HSCs. Using Tpo^DsRed-CreER knock-in mice, we showed that TPO is expressed by hepatocytes but not by bone marrow cells. Deletion of Tpo from hematopoietic cells, osteoblasts, or bone marrow mesenchymal stromal cells does not affect HSC number or function. However, when Tpo is deleted from hepatocytes, bone marrow HSCs are depleted. Thus, a cross-organ factor, circulating TPO made in the liver by hepatocytes, is required for bone marrow HSC maintenance. Our results demonstrate that systemic factors, in addition to the local niche, are a critical extrinsic component for HSC maintenance.

Fig. 1. TPO is expressed by hepatocytes but not by bone marrow cells

(A) qRT-PCR analysis of Tpo transcript levels (n = 3 mice; error bars indicate SD). (B) Schema of TPO expression analysis in Tpo^DsRed-CreER; loxpZsGreen mice. LSL, loxp-Stop-loxp. (C to J) Confocal images of liver sections from sham-treated (+Sham) or TMX-treated Tpo^DsRed-CreER; loxpZsGreen mice. DAPI, 4′,6-diamidino-2-phenylindole. (K to N) Confocal images of femur sections from sham- or TMX-treated Tpo^DsRed-CreER; loxpZsGreen mice.

Fig. 2. Osteoblasts and mesenchymal stromal cells are not critical sources of TPO for HSC maintenance

(A to C) Cellularity (A), HSC frequency (B), and bone marrow colony-forming cell frequency (C) were normal in Col2.3-cre;Tpo^fl/gfp mice (n = 4 to 5 mice). (D) Bone marrow cells (5 × 10⁵) from Col2.3-cre;Tpo^fl/gfp mice gave normal donor cell reconstitution compared to controls (in two experiments with a total of eight to nine recipient mice per genotype). (E to G) Cellularity (E), HSC frequency (F), and bone marrow colony-forming cell frequency (G) were normal in Lepr-cre;Tpo^fl/gfp mice (n = 5 to 6 mice). (H) Bone marrow cells (5 × 10⁵) from Lepr-cre;Tpo^fl/gfp mice gave normal levels of donor cell reconstitution compared to controls (in two experiments with a total of eight recipient mice per genotype). Controls (Con), Col2.3-cre;Tpo^+/gfp, Lepr-cre;Tpo^+/gfp, or Tpo^fl/gfp mice. Δ/gfp, Col2.3-cre;Tpo^fl/gfp or Lepr-cre;Tpo^fl/gfp mice. Data are means ± SD. ns, not significant (P > 0.10).

Fig. 3. Hepatocyte-derived TPO is required for HSC maintenance

(A to D) Confocal images of liver sections from Alb-cre;loxpTdTomato (Alb-cre;loxpTdT) mice. HNF4α, hepatocyte nuclear factor 4–α. (E) Platelet counts were decreased in Alb-cre;Tpo^fl/fl mice (n = 4). (F) Bone marrow CD41⁺ cell frequency was decreased in Alb-cre; Tpo^fl/fl mice (n = 5 to 7). (G) Cellularity was normal in the bone marrow and spleens from Alb-cre;Tpo^fl/fl mice (n = 5 to 6). (H) HSCs were depleted in the bone marrow but not in spleens from Alb-cre;Tpo^fl/fl mice (n = 6 to 8). (I and J) Colony-forming cell numbers were decreased in the bone marrow and trended lower in spleens (SPL) from Alb-cre; Tpo^fl/fl mice (n = 5 to 6). E, erythroid burst–forming unit; Mk, megakaryocyte; GM, granulocyte-macrophage; GEMM, granulocyte-erythrocyte-monocyte-megakaryocyte. (K) Bone marrow cells (5 × 10⁵) from Alb-cre;Tpo^fl/fl mice gave lower levels of donor cell reconstitution (in two experiments with a total of eight to nine recipient mice per genotype). (L) Recipient mice from (K) showed significant decrease in donor bone marrow HSC chimerism 16 weeks posttransplant (n = 6 mice per genotype). Con, Alb-cre;Tpo^+/+, Tpo^fl/fl, or Tpo^fl/+ mice; Δ/Δ, Alb-cre;Tpo^fl/fl mice. Data are means ± SD. ns, not significant (P > 0.10). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4. TPO from adult hepatocytes regulates bone marrow HSC maintenance and quiescence

(A to D) Confocal images of liver sections from AAV8-TBG-cre–treated loxpTdTomato mice. (E) Bone marrow and spleen cellularity was normal in Alb-cre;Tpo^fl/fl mice (n = 7 to 8). (F) Bone marrow HSCs were depleted in AAV8-TBG-cre–treated Tpo^fl/fl mice (n = 4 to 5). (G) Spleen HSC frequency tended to decrease in AAV8-TBG-cre–treated Tpo^fl/fl mice. (H and I) Bone marrow and spleen colony-forming cell frequencies decreased in AAV8-cre; Tpo^fl/fl mice (n = 6 to 7). (J) Bone marrow cells (5 × 10⁵) from AAV8-cre; Tpo^fl/fl mice gave lower levels of donor cell reconstitution than controls (in two to three experiments with a total of n = 16 for controls, n = 9 for 1 month after AAV8 treatment, and n = 14 for 2 months after AAV8 treatment). (K) Limit dilution assays showed a 6-fold reduction in functional HSCs in the bone marrow from AAV8-cre;Tpo^fl/fl mice compared with those in controls (in two independent experiments). (L) Annexin V⁺ DAPI⁻ bone marrow HSC frequency was not affected in AAV8-cre;Tpo^fl/fl mice (n = 5). (M) Bone marrow HSCs incorporated more bromodeoxyuridine (BrdU) in AAV8-cre; Tpo^fl/fl mice (n = 4). Con, Tpo^+/+ mice treated with AAV8-TBG-cre or Tpo^fl/fl mice treated with phosphate-buffered saline; Δ/Δ, Tpo^fl/fl mice treated with AAV8-TBG-cre. Data are means ± SD. ns, not significant (P > 0.10). *P < 0.05, **P < 0.01, ***P < 0.001.