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Biomechanical forces promote blood development through prostaglandin E2 and the cAMP-PKA signaling axis.

Diaz MF, Li N, Lee HJ, Adamo L, Evans SM, Willey HE, Arora N, Torisawa YS, Vickers DA, Morris SA, Naveiras O, Murthy SK, Ingber DE, Daley GQ, García-Cardeña G, Wenzel PL - J. Exp. Med. (2015)

Bottom Line: Furthermore, Ncx1 heartbeat mutants, as well as static cultures of AGM, exhibit lower levels of expression of prostaglandin synthases and reduced phosphorylation of the cAMP response element-binding protein (CREB).Similar to flow-exposed cultures, transient treatment of AGM with the synthetic analogue 16,16-dimethyl-PGE2 stimulates more robust engraftment of adult recipients and greater lymphoid reconstitution.These data provide one mechanism by which biomechanical forces induced by blood flow modulate hematopoietic potential.

View Article: PubMed Central - HTML - PubMed

Affiliation: Program in Children's Regenerative Medicine, Department of Pediatric Surgery, Center for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular Medicine, and Immunology Program, Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston, Houston, TX 77030 Program in Children's Regenerative Medicine, Department of Pediatric Surgery, Center for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular Medicine, and Immunology Program, Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston, Houston, TX 77030 Program in Children's Regenerative Medicine, Department of Pediatric Surgery, Center for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular Medicine, and Immunology Program, Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston, Houston, TX 77030.

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Long-term multilineage repopulation of the blood system is enhanced by WSS.Rag2−/− Il2rγ−/− recipients received 16 e.e. of E9.5 PSp cultured in the presence of either static (<0.0001 dyn/cm2) or WSS (5 dyn/cm2) conditions for 36 h. (A) Donor contribution to recipient leukocytes in peripheral blood is distinguishable as a discrete CD45.2+ CD45.1− population in WSS recipients (20-wk posttransplantation data shown). (B) Peripheral blood reconstitution is significantly greater from cells exposed to WSS (n = 17 static from six independent experiments, n = 15 WSS from seven independent experiments; two-way ANOVA: *, P = 0.03). (C) Representative flow cytometry plots from the WSS recipient in A show lineage+ engraftment from CD45.2+ donor–derived cells. (D) Lineages present in CD45.2+ donor peripheral blood from individual recipients at 5, 10, and 20 wk after transplantation. B lymphopoiesis and long-term multilineage potential are bolstered by WSS. (E) CD45.2+ donor PSps contribute to greater numbers of cells expressing B lineage markers during the posttransplant period (20 wk) when exposed to WSS (n = 17 static, n = 15 WSS; Mann–Whitney rank sum: *, P = 0.03). (F) Primary recipient bone marrow was analyzed at 20 wk after transplantation of cultured PSp with markers of B lineage maturation (two static and three WSS recipients are shown alongside adult donor strain). B cells within the bone marrow collected from WSS recipients resemble adult donor marrow. (G) Production of early and late B lineages in primary bone marrow was discriminated by B220 and CD43 cell surface expression in the CD45.2+ donor population. Early B lineage phenotypes were marginally elevated by WSS (B220+ CD43+: n = 3 static, n = 5 WSS; unpaired Student’s t test: *, P = 0.13). Data are represented as mean ± SEM.
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fig2: Long-term multilineage repopulation of the blood system is enhanced by WSS.Rag2−/− Il2rγ−/− recipients received 16 e.e. of E9.5 PSp cultured in the presence of either static (<0.0001 dyn/cm2) or WSS (5 dyn/cm2) conditions for 36 h. (A) Donor contribution to recipient leukocytes in peripheral blood is distinguishable as a discrete CD45.2+ CD45.1− population in WSS recipients (20-wk posttransplantation data shown). (B) Peripheral blood reconstitution is significantly greater from cells exposed to WSS (n = 17 static from six independent experiments, n = 15 WSS from seven independent experiments; two-way ANOVA: *, P = 0.03). (C) Representative flow cytometry plots from the WSS recipient in A show lineage+ engraftment from CD45.2+ donor–derived cells. (D) Lineages present in CD45.2+ donor peripheral blood from individual recipients at 5, 10, and 20 wk after transplantation. B lymphopoiesis and long-term multilineage potential are bolstered by WSS. (E) CD45.2+ donor PSps contribute to greater numbers of cells expressing B lineage markers during the posttransplant period (20 wk) when exposed to WSS (n = 17 static, n = 15 WSS; Mann–Whitney rank sum: *, P = 0.03). (F) Primary recipient bone marrow was analyzed at 20 wk after transplantation of cultured PSp with markers of B lineage maturation (two static and three WSS recipients are shown alongside adult donor strain). B cells within the bone marrow collected from WSS recipients resemble adult donor marrow. (G) Production of early and late B lineages in primary bone marrow was discriminated by B220 and CD43 cell surface expression in the CD45.2+ donor population. Early B lineage phenotypes were marginally elevated by WSS (B220+ CD43+: n = 3 static, n = 5 WSS; unpaired Student’s t test: *, P = 0.13). Data are represented as mean ± SEM.

Mentions: E9.5 PSp has previously been found to produce primarily B1a lymphocyte progenitors when delivered into immunocompromised neonatal recipients (Yoshimoto et al., 2011) or intact under the adult kidney capsule (Godin et al., 1993) and requires whole organ culture for several days to acquire long-term multilineage repopulating activity in vivo (Cumano et al., 2001). To evaluate whether changes induced by WSS were capable of producing greater functional competence in stem and progenitor cells, we transplanted dissociated PSp from E9.5 embryos (21–29 somite pairs) cultured with or without WSS for 36 h into irradiated adult Rag2−/− Il2rγ−/− recipients. Based upon the expectation that dissociated E9.5 PSp would provide absent to scant engraftment of adult recipients long term (Godin et al., 1993; Cumano et al., 2001), we transplanted each recipient with 16 embryo equivalents (e.e.). We detected low-level engraftment in the peripheral blood of 4 of 17 animals injected with E9.5 PSp cultured under static conditions, which dropped further by 10 wk in the surviving 10 animals, suggesting that the majority of engrafting cells were exhaustible short-term progenitors (Fig. 2, A and B). At 20 wk, static cultures contributed to <1.4% of total peripheral blood leukocytes in all but one recipient (2.8% CD45.2+). In contrast, WSS produced higher initial engraftment levels in 6 of 15 recipients, which persisted long term in 5 animals (Fig. 2 B). PSp contributed to major adult blood lineages, including B lymphoid (B220, CD19, and IgM), T lymphoid (CD3, CD4, and CD8), and myeloid (Mac1 and Gr1) populations (Fig. 2 C). WSS exposure promoted earlier emergence of B lymphocytes and sustained B lymphopoiesis in the periphery beyond 20 wk (Fig. 2, D and E). Greater B cell maturity was also apparent in the bone marrow (Fig. 2 F). Specifically, greater numbers of cells were in stages of pre-pro- and pro-B commitment (B220+ CD43+), with modest increases in late-stage pre-B and B development (B220+ CD43−) typical of adult-type bone marrow progenitors (Fig. 2 G; Hardy and Hayakawa, 1991). CD45.2+ bone marrow cells were subsequently transferred to secondary recipients and found to reconstitute four of seven WSS and two of five static recipients (Fig. 3, A and B). Importantly, peak chimerism was substantially higher for three of the WSS donors (4.2%, 12.2%, and 61.3%) as compared with static donors (0.9% and 2.1%). B and T cells were detectable as discrete populations and persisted long term in WSS recipients (Fig. 3, C and D). Collectively, these data suggest that WSS serves as a critical developmental signal that promotes commitment of precursors to the blood lineage and endows nascent HSCs and progenitors with the functional competence necessary to engraft the adult hematopoietic niche.


Biomechanical forces promote blood development through prostaglandin E2 and the cAMP-PKA signaling axis.

Diaz MF, Li N, Lee HJ, Adamo L, Evans SM, Willey HE, Arora N, Torisawa YS, Vickers DA, Morris SA, Naveiras O, Murthy SK, Ingber DE, Daley GQ, García-Cardeña G, Wenzel PL - J. Exp. Med. (2015)

Long-term multilineage repopulation of the blood system is enhanced by WSS.Rag2−/− Il2rγ−/− recipients received 16 e.e. of E9.5 PSp cultured in the presence of either static (<0.0001 dyn/cm2) or WSS (5 dyn/cm2) conditions for 36 h. (A) Donor contribution to recipient leukocytes in peripheral blood is distinguishable as a discrete CD45.2+ CD45.1− population in WSS recipients (20-wk posttransplantation data shown). (B) Peripheral blood reconstitution is significantly greater from cells exposed to WSS (n = 17 static from six independent experiments, n = 15 WSS from seven independent experiments; two-way ANOVA: *, P = 0.03). (C) Representative flow cytometry plots from the WSS recipient in A show lineage+ engraftment from CD45.2+ donor–derived cells. (D) Lineages present in CD45.2+ donor peripheral blood from individual recipients at 5, 10, and 20 wk after transplantation. B lymphopoiesis and long-term multilineage potential are bolstered by WSS. (E) CD45.2+ donor PSps contribute to greater numbers of cells expressing B lineage markers during the posttransplant period (20 wk) when exposed to WSS (n = 17 static, n = 15 WSS; Mann–Whitney rank sum: *, P = 0.03). (F) Primary recipient bone marrow was analyzed at 20 wk after transplantation of cultured PSp with markers of B lineage maturation (two static and three WSS recipients are shown alongside adult donor strain). B cells within the bone marrow collected from WSS recipients resemble adult donor marrow. (G) Production of early and late B lineages in primary bone marrow was discriminated by B220 and CD43 cell surface expression in the CD45.2+ donor population. Early B lineage phenotypes were marginally elevated by WSS (B220+ CD43+: n = 3 static, n = 5 WSS; unpaired Student’s t test: *, P = 0.13). Data are represented as mean ± SEM.
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fig2: Long-term multilineage repopulation of the blood system is enhanced by WSS.Rag2−/− Il2rγ−/− recipients received 16 e.e. of E9.5 PSp cultured in the presence of either static (<0.0001 dyn/cm2) or WSS (5 dyn/cm2) conditions for 36 h. (A) Donor contribution to recipient leukocytes in peripheral blood is distinguishable as a discrete CD45.2+ CD45.1− population in WSS recipients (20-wk posttransplantation data shown). (B) Peripheral blood reconstitution is significantly greater from cells exposed to WSS (n = 17 static from six independent experiments, n = 15 WSS from seven independent experiments; two-way ANOVA: *, P = 0.03). (C) Representative flow cytometry plots from the WSS recipient in A show lineage+ engraftment from CD45.2+ donor–derived cells. (D) Lineages present in CD45.2+ donor peripheral blood from individual recipients at 5, 10, and 20 wk after transplantation. B lymphopoiesis and long-term multilineage potential are bolstered by WSS. (E) CD45.2+ donor PSps contribute to greater numbers of cells expressing B lineage markers during the posttransplant period (20 wk) when exposed to WSS (n = 17 static, n = 15 WSS; Mann–Whitney rank sum: *, P = 0.03). (F) Primary recipient bone marrow was analyzed at 20 wk after transplantation of cultured PSp with markers of B lineage maturation (two static and three WSS recipients are shown alongside adult donor strain). B cells within the bone marrow collected from WSS recipients resemble adult donor marrow. (G) Production of early and late B lineages in primary bone marrow was discriminated by B220 and CD43 cell surface expression in the CD45.2+ donor population. Early B lineage phenotypes were marginally elevated by WSS (B220+ CD43+: n = 3 static, n = 5 WSS; unpaired Student’s t test: *, P = 0.13). Data are represented as mean ± SEM.
Mentions: E9.5 PSp has previously been found to produce primarily B1a lymphocyte progenitors when delivered into immunocompromised neonatal recipients (Yoshimoto et al., 2011) or intact under the adult kidney capsule (Godin et al., 1993) and requires whole organ culture for several days to acquire long-term multilineage repopulating activity in vivo (Cumano et al., 2001). To evaluate whether changes induced by WSS were capable of producing greater functional competence in stem and progenitor cells, we transplanted dissociated PSp from E9.5 embryos (21–29 somite pairs) cultured with or without WSS for 36 h into irradiated adult Rag2−/− Il2rγ−/− recipients. Based upon the expectation that dissociated E9.5 PSp would provide absent to scant engraftment of adult recipients long term (Godin et al., 1993; Cumano et al., 2001), we transplanted each recipient with 16 embryo equivalents (e.e.). We detected low-level engraftment in the peripheral blood of 4 of 17 animals injected with E9.5 PSp cultured under static conditions, which dropped further by 10 wk in the surviving 10 animals, suggesting that the majority of engrafting cells were exhaustible short-term progenitors (Fig. 2, A and B). At 20 wk, static cultures contributed to <1.4% of total peripheral blood leukocytes in all but one recipient (2.8% CD45.2+). In contrast, WSS produced higher initial engraftment levels in 6 of 15 recipients, which persisted long term in 5 animals (Fig. 2 B). PSp contributed to major adult blood lineages, including B lymphoid (B220, CD19, and IgM), T lymphoid (CD3, CD4, and CD8), and myeloid (Mac1 and Gr1) populations (Fig. 2 C). WSS exposure promoted earlier emergence of B lymphocytes and sustained B lymphopoiesis in the periphery beyond 20 wk (Fig. 2, D and E). Greater B cell maturity was also apparent in the bone marrow (Fig. 2 F). Specifically, greater numbers of cells were in stages of pre-pro- and pro-B commitment (B220+ CD43+), with modest increases in late-stage pre-B and B development (B220+ CD43−) typical of adult-type bone marrow progenitors (Fig. 2 G; Hardy and Hayakawa, 1991). CD45.2+ bone marrow cells were subsequently transferred to secondary recipients and found to reconstitute four of seven WSS and two of five static recipients (Fig. 3, A and B). Importantly, peak chimerism was substantially higher for three of the WSS donors (4.2%, 12.2%, and 61.3%) as compared with static donors (0.9% and 2.1%). B and T cells were detectable as discrete populations and persisted long term in WSS recipients (Fig. 3, C and D). Collectively, these data suggest that WSS serves as a critical developmental signal that promotes commitment of precursors to the blood lineage and endows nascent HSCs and progenitors with the functional competence necessary to engraft the adult hematopoietic niche.

Bottom Line: Furthermore, Ncx1 heartbeat mutants, as well as static cultures of AGM, exhibit lower levels of expression of prostaglandin synthases and reduced phosphorylation of the cAMP response element-binding protein (CREB).Similar to flow-exposed cultures, transient treatment of AGM with the synthetic analogue 16,16-dimethyl-PGE2 stimulates more robust engraftment of adult recipients and greater lymphoid reconstitution.These data provide one mechanism by which biomechanical forces induced by blood flow modulate hematopoietic potential.

View Article: PubMed Central - HTML - PubMed

Affiliation: Program in Children's Regenerative Medicine, Department of Pediatric Surgery, Center for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular Medicine, and Immunology Program, Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston, Houston, TX 77030 Program in Children's Regenerative Medicine, Department of Pediatric Surgery, Center for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular Medicine, and Immunology Program, Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston, Houston, TX 77030 Program in Children's Regenerative Medicine, Department of Pediatric Surgery, Center for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular Medicine, and Immunology Program, Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston, Houston, TX 77030.

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Related in: MedlinePlus