Cell-Cell Contact and Anatomical Compatibility in Stromal Cell-Mediated HSC Support During Development
http://www.100md.com
《干细胞学杂志》
Department of Cell Biology and Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands
Key Words. Stromal microenvironment ? Hematopoietic stem cells ? Embryo ? AGM ? Transwell ? Cell-cell contact
Elaine Dzierzak, Ph.D., Erasmus University Medical Center, Dept. of Cell Biology and Genetics, P. O. Box 1738, 3000 DR Rotterdam, The Netherlands. Telephone: 31-10-408-7172; Fax: 31-10-408-9468; e-mail: e.dzierzak@erasmusmc.nl
ABSTRACT
In the adult, mature blood cells are derived from hematopoietic stem cells (HSCs) through a complex cell differentiation hierarchy. HSCs are defined by the ability to self-renew and differentiate to all blood lineages, as demonstrated by clonal marking and complete repopulation of hematopoietic ablated adult recipient mice . The potential of HSCs is maintained primarily by the microenvironment, which consists of stromal cells (SCs). The experiments of Dexter originally showed the importance of SCs, with colony-forming unit spleen (CFU-S) ability being lost in primary bone marrow (BM) cultures, when no SCs were present . Since then other studies have shown the close association of HSCs and SCs in vitro with embryonic cells and in vivo within niches of the adult BM and also the importance of cell-cell contact in long-term culture-initiating cells (LTC-IC) and cobblestone area-forming cell ex vivo cultures .
Although HSCs are harbored in the BM microenvironment of the adult mouse, during ontogeny HSCs are found in different anatomical sites. These sites include first the aorta-gonads-mesonephros (AGM) region and then the yolk sac (YS) and fetal liver (FL) . It is thought that these microenvironments differ in their capacity to support hematopoietic progenitors and/or HSCs and also that ontogenically early HSCs may possess different characteristics allowing them to be harbored and active in the various embryonic hematopoietic microenvironments. Indeed, it has been shown that while FL hematopoietic progenitors (BFU-E) are not supported in vitro in a BM stromal microenvironment , both adult BM and FL HSCs can interact effectively in vitro with the FL stromal microenvironment and in vivo by yielding complete hematopoietic repopulation in irradiated adult recipients. Since the first HSCs emerge during mouse midgestation in the AGM region and AGM explant cultures show large increases in HSC numbers, this microenvironment and the HSCs within it are especially interesting for our understanding of hematopoietic regulation.
To study the microenvironment, SC lines from the mouse AGM region and its component subregions, aorta (Ao) and urogenital ridges (UG), have been generated . AGM-derived stromal clones have been shown to maintain HSCs from different developmental, anatomical, and species sources such as human umbilical cord blood (UCB) , mouse FL , mouse BM, and sorted CD34+ c-kit+ populations of embryonic day (E)11 YS/AGM . However, none of these studies have directly compared the quality of HSC support provided by AGM SCs to midgestation aortic, UG or YS HSCs, the three most closely related HSC populations during this stage of development . Moreover, since these embryonic cells are tightly organized and undergoing proliferation and differentiation in the context of local signaling factors, it is postulated that cell-cell contact between HSCs and stroma is important in the AGM.
Previous studies by several laboratories have shown that physical contact between human hematopoietic cells from fetal/adult sources and mouse SCs is important for providing the efficient maintenance of human progenitors and HSCs . The mouse stromal line (MS5) provides better support of human progenitors than human BM stroma, suggesting that the match between species is not important for adult hematopoietic cells. Others have found efficient maintenance of human hematopoietic progenitors/HSCs in non-contact cultures or in the complete absence of SCs over periods of 2 to 5 weeks . While the conditions necessary for the support of human hematopoietic cells remain controversial, to date little is known about the stromal contact requirements for mouse HSCs from the midgestational hematopoietic sites. Studies with FL hematopoietic progenitors suggest that contact with AGM stromal lines is essential . However, these studies did not address whether contact is advantageous for specific HSCs from the same or different developmental/anatomical site(s), particularly the AGM region. Thus, we studied the cell-cell contact requirements of HSCs from three different developmentally early anatomical hematopoietic sites in UG stromal clone co-cultures. We show here the highest support of E11 Ao HSCs in such co-cultures as compared to HSCs from the E11 UG and YS and that contact is required. These results indicate differences in the interactions between HSCs from different sources and the stroma, and suggest an important role for AGM microenvironment in the maintenance and expansion of the earliest Ao-derived HSCs.
MATERIALS AND METHODS
Three SC lines previously isolated from the UGs of E11 AGMs were chosen for these studies: UG 26.1B6 (1B6) because it is the best supporter for mouse BM HSCs in long-term co-cultures , UG 26.3D4 (3D4) since it most highly supports human UCB hematopoietic progenitors in long-term co-cultures , and UG 26.3B5 (3B5) as a non-supportive stromal control, since it has been shown to be unsupportive of mouse BM LTC-ICs, resulting in 35-fold fewer CFU-cultures (CFU-C) as compared to 1B6 and 3D4 .
UG Stromal Co-Cultures Support Midgestation Aortic Hematopoietic Progenitors More Efficiently in Contact Cultures
To determine if the three stromal clones 1B6, 3D4, and 3B5 could support E11 aortic hematopoietic progenitors, conventional co-cultures were established in which contact could occur between the hematopoietic cells and the SCs. After 5 days, Ao cells were tested for hematopoietic progenitor activity. As shown in Figure 1, most CFU-Cs were found in the adherent fractions (300 to 500 CFU-Cs/E11 Ao) and fewer in the non-adherent fractions (40 to 300 CFU-Cs/E11 Ao) of the co-cultures. CFU-Cs varied in number between the stromal clones, but not in size or morphology. Indeed, in all cases CFU-granulocyte macrophage, BFU-E, and CFU-mix were found. No CFU-Cs were found in the absence of stroma. The 1B6 and 3D4 stromal clones provided similarly good support for midgestation Ao hematopoietic progenitors and expanded input numbers by 9- to 14-fold. However, while 3B5 did not support progenitors in the non-adherent fraction, it did unexpectedly support hematopoietic progenitors in the adherent fraction, increasing input progenitor numbers by 10-fold. Thus, 3B5 can no longer be considered as a non-supportive stromal clone. Nonetheless, it has very different supportive properties than 1B6 and 3D4. The differences most likely are inherent in signaling interactions and/or adhesive properties.
Figure 1. Hematopoietic progenitors are maintained in contact and non-contact cultures with midgestation UG stromal clones. A pool of E11 Ao cells was obtained. The input number of aortic hematopoietic progenitors was measured by MC CFU assay and found to be 5.2 ± 1.6 per 0.1 (ee) of tissue. For contact cultures, a single cell suspension of Ao cells was plated at 1 ee of cells on each of the UG 26 SC lines (irradiated at 30 Gy) and cultured for 5 days at 33°C. The UG 26 SC lines are as indicated: 1B6, 3D4, and 3B5. No line indicates the results of the control culture of Ao cells in which no SC line was used. For non-contact cultures, a single cell suspension of Ao cells was cultured in the transwell above the SCs indicated. All cells were harvested from the transwell of the non-contact cultures. From the contact cultures, the non-adherent and adherent cell fractions (containing the stromal line) were harvested. Cells were plated in MC 37°C for 7 days and hematopoietic colonies counted (CFU-GM, BFU-E, CFU-mix). The mean of triplicate samples of four to six experiments (except for contact adherent data, n = 2) is indicated above each bar along with the standard error.
Next we compared the support of CFU-Cs in conventional stromal co-cultures with the support provided by transwell co-cultures (non-contact). In such transwell co-cultures the SCs are physically separated from the hematopoietic cells by a filter, which is permeable to growth factors/signaling molecules. A clear decrease in CFU-C numbers is observed in non-contact co-cultures of all three stromal clones. The cumulative numbers of CFU-Cs were decreased by factors of 6-, 5-, and 5-fold for 1B6, 3D4, and 3B5, respectively, in non-contact cultures, as compared to contact cultures. Finally, while the CFU-C numbers in contact co-cultures are consistent with a 9- to 14-fold expansion of the input number of E11 Ao progenitor cells (5.2 ± 1.6), the non-contact cultures showed only a 1.5- to 3-fold expansion. These data demonstrate that all three E11 UG-derived stromal clones can maintain E11 Ao CFU-Cs independent of cell-cell interactions, but that direct contact is required for efficient expansion of CFU-Cs.
Contact with UG Stromal Clones is Essential to Maintain and Expand Midgestation Aortic HSCs
We next examined the effects of cell-cell contact on the maintenance and expansion of HSCs from E11 Aos, UGs, and YSs in co-cultures with the three UG stromal clones. After contact or non-contact co-culture of genetically marked embryonic cells with the stromal lines, cells were transplanted into irradiated adult recipients and tested for HSC repopulation 4 months post-transplantation. Also, 1 ee of these cells was injected directly into irradiated recipients as a control for input HSCs in the co-cultures. In the long-term in vivo repopulation assay, we found that all three stromal lines behaved similarly in their support of HSCs. As shown in Table 1, 4/9, 5/9, and 2/5 recipients were high level, multilineage repopulated with a limiting dilution (0.3 ee) of Ao cells co-cultured with 1B6, 3D4, and 3B5 respectively. Thus, these contact co-cultures support a 3-fold increase in Ao HSCs as compared to input HSC number. Moreover, despite injection of 1 ee of cultured Ao cells, no Ao HSCs were found in non-contact stromal co-cultures (0/4, 0/3, and 0/5 recipients repopulated/transplanted for 1B6, 3D4, and 3B5, respectively, or 0/12 when data are pooled). Hence, E11 Ao-derived HSCs require SC contact for maintenance and expansion ex vivo.
Table 1. Support of HSCs from different embryonic sources in contact and non-contact cultures with midgestation UG stromal clones
To directly compare support of HSCs from other E11 hematopoietic tissues to Ao HSCs, and to determine if SC contact is required for the support of these HSCs, contact and non-contact co-cultures of 1B6, 3D4 and 3B5 were established with UG, YS and FL cells. Injection of a limiting dilution (0.3 ee) of UG cells from contact co-cultures with 3D4 revealed the presence of UG HSCs in one out of six recipients, while injection of 1 ee of uncultured UG cells gave no repopulation in five recipients. This increase in UG HSC activity requires stromal contact, since no UG HSCs were found after 1 ee of such cells was transplanted. In contrast to the increase found for Ao and UG HSCs in contact co-cultures, no recipients were found repopulated after injection of 0.3 ee of contact co-cultured YS cells. YS HSC activity was also absent in the non-contact cultures as determined by transplantation of 1 ee of cells. In studies with FL cells, support of HSCs was found in contact cultures but not in non-contact cultures (data not shown). Hence, UG SC lines expand and maintain Ao and UG HSCs, but do not support YS HSCs. These results suggest that HSCs and SCs are most functionally compatible when derived from closely related embryonic tissue. Moreover, contact with UG stromal clones is essential for the support/expansion of these midgestation HSCs.
DISCUSSION
We thank all laboratory members for their helpful comments and assistance with various aspects of this work. We especially thank Robert Oostendorp, Rob Ploemacher, Trui Visser, Kam-Wing Ling, Katrin Ottersbach, Marian Peeters and Karim Hussein and the Erasmus Dierexperimenteel Centrum for animal care. We appreciate the critical review of the manuscript by Dr. Catherine Robin. This work was supported by the NIH RO DK51077 and ErasmusMC Breedtestrategie Program.
REFERENCES
Lemischka IR, Raulet DH, Mulligan RC et al. Developmental potential and dynamic behavior of hematopoietic stem cells. Cell 1986;45:917–927.
Dexter TM, Testa NG. Differentiation and proliferation of hemopoietic cells in culture. Methods Cell Biol 1976;14:387–405.
Young PE, Baumhueter S, Lasky LA et al. The sialomucin CD34 is expressed on hematopoietic cells and blood vessels during murine development. Blood 1995;85:96–105.
Tavian M, Coulombel L, Luton D et al. Aorta-associated CD34+ hematopoietic cells in the early human embryo. Blood 1996;87:67–72.
Roy V, Verfaillie CM. Soluble factor(s) produced by adult bone marrow stroma inhibit in vitro proliferation and differentiation of fetal liver BFU-E by inducing apoptosis. J Clin Invest 1997;100:912–920.
Moore KA, Ema H, Lemischka IR et al. In vitro maintenance of highly purified, transplantable hematopoietic stem cells. Blood 1997;89:4337–4347.
Coulombel L, Eaves AC, Eaves CJ et al. Enzymatic treatment of long-term human marrow cultures reveals the preferential location of primitive hemopoietic progenitors in the adherent layer. Blood 1983;62:291–297.
Breems DA, Blokland EA, Siebel KE et al. Stroma-contact prevents loss of hematopoietic stem cell quality during ex vivo expansion of CD34+ mobilized peripheral blood stem cells. Blood 1998;91:111–117.
Dzierzak E. Hematopoietic stem cells and their precursors: developmental diversity and lineage relationships. Immunol Rev 2002;187:126–138.
Muller AM, Medvinsky A, Strouboulis J et al. Development of hematopoietic stem cell activity in the mouse embryo. Immunity 1994;1:291–301.
Medvinsky A, Dzierzak E. Definitive hematopoiesis is autonomously initiated by the AGM region. Cell 1996;86:897–906.
Ohkawara JI, Ikebuchi K, Fujihara M et al. Culture system for extensive production of CD19+IgM+ cells by human cord blood CD34+ progenitors. Leukemia 1998;12:764–771.
Ohneda O, Fennie C, Zheng Z et al. Hematopoietic stem cell maintenance and differentiation are supported by embryonic aorta-gonad-mesonephros region-derived endothelium. Blood 1998;92:908–919.
Xu MJ, Tsuji K, Ueda T et al. Stimulation of mouse and human primitive hematopoiesis by murine embryonic aorta-gonad-mesonephros-derived stromal cell lines. Blood 1998;92:2032–2040.
Oostendorp RA, Harvey KN, Kusadasi N et al. Stromal cell lines from mouse aorta-gonads-mesonephros subregions are potent supporters of hematopoietic stem cell activity. Blood 2002;99:1183–1189.
Oostendorp RA, Medinsky AJ, Kusadasi N et al. Embryonal subregion-derived stromal cell lines from novel temperature-sensitive SV40 T antigen transgenic mice support hematopoiesis. J Cell Sci 2002;115:2099–2108.
Kusadasi N, Oostendorp RA, Koevoet WJ et al. Stromal cells from murine embryonic aorta-gonad-mesonephros region, liver and gut mesentery expand human umbilical cord blood-derived CAFC(week 6) in extended long-term cultures. Leukemia 2002;16:1782–1790.
Bennaceur-Griscelli A, Tourino C, Izac B et al. Murine stromal cells counteract the loss of long-term culture-initiating cell potential induced by cytokines in CD34(+)CD38(low/neg) human bone marrow cells. Blood 1999;94:529–538.
Lewis ID, Almeida-Porada G, Du J et al. Umbilical cord blood cells capable of engrafting in primary, secondary, and tertiary xenogeneic hosts are preserved after ex vivo culture in a noncontact system. Blood 2001;97:3441–3449.
Gupta P, Oegema TR Jr, Brazil JJ et al. Human LTC-IC can be maintained for at least 5 weeks in vitro when interleukin-3 and a single chemokine are combined with O-sulfated heparan sulfates: requirement for optimal binding interactions of heparan sulfate with early-acting cytokines and matrix proteins. Blood 2000;95:147–155.
Strouboulis J, Dillon N, Grosveld F et al. Developmental regulation of a complete 70-kb human beta-globin locus in transgenic mice. Genes Dev 1992;6:1857–1864.
Ma X, Robin C, Ottersbach K et al. The Ly-6A (Sca-1) GFP transgene is expressed in all adult mouse hematopoietic stem cells. STEM CELLS 2002;20:514–521.
de Bruijn MF, Ma X, Robin C et al. Hematopoietic stem cells localize to the endothelial cell layer in the midgestation mouse aorta. Immunity 2002;16:673–683.
Roy V, Verfaillie CM. Expression and function of cell adhesion molecules on fetal liver, cord blood and bone marrow hematopoietic progenitors: implications for anatomical localization and developmental stage specific regulation of hematopoiesis. Exp Hematol 1999;27:302–312.
de Bruijn MF, Speck NA, Peeters MC et al. Definitive hematopoietic stem cells first develop within the major arterial regions of the mouse embryo. EMBO J 2000;19:2465–2474.
Takeuchi M, Sekiguchi T, Hara T et al. Cultivation of aorta-gonad-mesonephros-derived hematopoietic stem cells in the fetal liver microenvironment amplifies long-term repopulating activity and enhances engraftment to the bone marrow. Blood 2002;99:1190–1196.(Kirsty Harvey, Elaine Dzi)
Key Words. Stromal microenvironment ? Hematopoietic stem cells ? Embryo ? AGM ? Transwell ? Cell-cell contact
Elaine Dzierzak, Ph.D., Erasmus University Medical Center, Dept. of Cell Biology and Genetics, P. O. Box 1738, 3000 DR Rotterdam, The Netherlands. Telephone: 31-10-408-7172; Fax: 31-10-408-9468; e-mail: e.dzierzak@erasmusmc.nl
ABSTRACT
In the adult, mature blood cells are derived from hematopoietic stem cells (HSCs) through a complex cell differentiation hierarchy. HSCs are defined by the ability to self-renew and differentiate to all blood lineages, as demonstrated by clonal marking and complete repopulation of hematopoietic ablated adult recipient mice . The potential of HSCs is maintained primarily by the microenvironment, which consists of stromal cells (SCs). The experiments of Dexter originally showed the importance of SCs, with colony-forming unit spleen (CFU-S) ability being lost in primary bone marrow (BM) cultures, when no SCs were present . Since then other studies have shown the close association of HSCs and SCs in vitro with embryonic cells and in vivo within niches of the adult BM and also the importance of cell-cell contact in long-term culture-initiating cells (LTC-IC) and cobblestone area-forming cell ex vivo cultures .
Although HSCs are harbored in the BM microenvironment of the adult mouse, during ontogeny HSCs are found in different anatomical sites. These sites include first the aorta-gonads-mesonephros (AGM) region and then the yolk sac (YS) and fetal liver (FL) . It is thought that these microenvironments differ in their capacity to support hematopoietic progenitors and/or HSCs and also that ontogenically early HSCs may possess different characteristics allowing them to be harbored and active in the various embryonic hematopoietic microenvironments. Indeed, it has been shown that while FL hematopoietic progenitors (BFU-E) are not supported in vitro in a BM stromal microenvironment , both adult BM and FL HSCs can interact effectively in vitro with the FL stromal microenvironment and in vivo by yielding complete hematopoietic repopulation in irradiated adult recipients. Since the first HSCs emerge during mouse midgestation in the AGM region and AGM explant cultures show large increases in HSC numbers, this microenvironment and the HSCs within it are especially interesting for our understanding of hematopoietic regulation.
To study the microenvironment, SC lines from the mouse AGM region and its component subregions, aorta (Ao) and urogenital ridges (UG), have been generated . AGM-derived stromal clones have been shown to maintain HSCs from different developmental, anatomical, and species sources such as human umbilical cord blood (UCB) , mouse FL , mouse BM, and sorted CD34+ c-kit+ populations of embryonic day (E)11 YS/AGM . However, none of these studies have directly compared the quality of HSC support provided by AGM SCs to midgestation aortic, UG or YS HSCs, the three most closely related HSC populations during this stage of development . Moreover, since these embryonic cells are tightly organized and undergoing proliferation and differentiation in the context of local signaling factors, it is postulated that cell-cell contact between HSCs and stroma is important in the AGM.
Previous studies by several laboratories have shown that physical contact between human hematopoietic cells from fetal/adult sources and mouse SCs is important for providing the efficient maintenance of human progenitors and HSCs . The mouse stromal line (MS5) provides better support of human progenitors than human BM stroma, suggesting that the match between species is not important for adult hematopoietic cells. Others have found efficient maintenance of human hematopoietic progenitors/HSCs in non-contact cultures or in the complete absence of SCs over periods of 2 to 5 weeks . While the conditions necessary for the support of human hematopoietic cells remain controversial, to date little is known about the stromal contact requirements for mouse HSCs from the midgestational hematopoietic sites. Studies with FL hematopoietic progenitors suggest that contact with AGM stromal lines is essential . However, these studies did not address whether contact is advantageous for specific HSCs from the same or different developmental/anatomical site(s), particularly the AGM region. Thus, we studied the cell-cell contact requirements of HSCs from three different developmentally early anatomical hematopoietic sites in UG stromal clone co-cultures. We show here the highest support of E11 Ao HSCs in such co-cultures as compared to HSCs from the E11 UG and YS and that contact is required. These results indicate differences in the interactions between HSCs from different sources and the stroma, and suggest an important role for AGM microenvironment in the maintenance and expansion of the earliest Ao-derived HSCs.
MATERIALS AND METHODS
Three SC lines previously isolated from the UGs of E11 AGMs were chosen for these studies: UG 26.1B6 (1B6) because it is the best supporter for mouse BM HSCs in long-term co-cultures , UG 26.3D4 (3D4) since it most highly supports human UCB hematopoietic progenitors in long-term co-cultures , and UG 26.3B5 (3B5) as a non-supportive stromal control, since it has been shown to be unsupportive of mouse BM LTC-ICs, resulting in 35-fold fewer CFU-cultures (CFU-C) as compared to 1B6 and 3D4 .
UG Stromal Co-Cultures Support Midgestation Aortic Hematopoietic Progenitors More Efficiently in Contact Cultures
To determine if the three stromal clones 1B6, 3D4, and 3B5 could support E11 aortic hematopoietic progenitors, conventional co-cultures were established in which contact could occur between the hematopoietic cells and the SCs. After 5 days, Ao cells were tested for hematopoietic progenitor activity. As shown in Figure 1, most CFU-Cs were found in the adherent fractions (300 to 500 CFU-Cs/E11 Ao) and fewer in the non-adherent fractions (40 to 300 CFU-Cs/E11 Ao) of the co-cultures. CFU-Cs varied in number between the stromal clones, but not in size or morphology. Indeed, in all cases CFU-granulocyte macrophage, BFU-E, and CFU-mix were found. No CFU-Cs were found in the absence of stroma. The 1B6 and 3D4 stromal clones provided similarly good support for midgestation Ao hematopoietic progenitors and expanded input numbers by 9- to 14-fold. However, while 3B5 did not support progenitors in the non-adherent fraction, it did unexpectedly support hematopoietic progenitors in the adherent fraction, increasing input progenitor numbers by 10-fold. Thus, 3B5 can no longer be considered as a non-supportive stromal clone. Nonetheless, it has very different supportive properties than 1B6 and 3D4. The differences most likely are inherent in signaling interactions and/or adhesive properties.
Figure 1. Hematopoietic progenitors are maintained in contact and non-contact cultures with midgestation UG stromal clones. A pool of E11 Ao cells was obtained. The input number of aortic hematopoietic progenitors was measured by MC CFU assay and found to be 5.2 ± 1.6 per 0.1 (ee) of tissue. For contact cultures, a single cell suspension of Ao cells was plated at 1 ee of cells on each of the UG 26 SC lines (irradiated at 30 Gy) and cultured for 5 days at 33°C. The UG 26 SC lines are as indicated: 1B6, 3D4, and 3B5. No line indicates the results of the control culture of Ao cells in which no SC line was used. For non-contact cultures, a single cell suspension of Ao cells was cultured in the transwell above the SCs indicated. All cells were harvested from the transwell of the non-contact cultures. From the contact cultures, the non-adherent and adherent cell fractions (containing the stromal line) were harvested. Cells were plated in MC 37°C for 7 days and hematopoietic colonies counted (CFU-GM, BFU-E, CFU-mix). The mean of triplicate samples of four to six experiments (except for contact adherent data, n = 2) is indicated above each bar along with the standard error.
Next we compared the support of CFU-Cs in conventional stromal co-cultures with the support provided by transwell co-cultures (non-contact). In such transwell co-cultures the SCs are physically separated from the hematopoietic cells by a filter, which is permeable to growth factors/signaling molecules. A clear decrease in CFU-C numbers is observed in non-contact co-cultures of all three stromal clones. The cumulative numbers of CFU-Cs were decreased by factors of 6-, 5-, and 5-fold for 1B6, 3D4, and 3B5, respectively, in non-contact cultures, as compared to contact cultures. Finally, while the CFU-C numbers in contact co-cultures are consistent with a 9- to 14-fold expansion of the input number of E11 Ao progenitor cells (5.2 ± 1.6), the non-contact cultures showed only a 1.5- to 3-fold expansion. These data demonstrate that all three E11 UG-derived stromal clones can maintain E11 Ao CFU-Cs independent of cell-cell interactions, but that direct contact is required for efficient expansion of CFU-Cs.
Contact with UG Stromal Clones is Essential to Maintain and Expand Midgestation Aortic HSCs
We next examined the effects of cell-cell contact on the maintenance and expansion of HSCs from E11 Aos, UGs, and YSs in co-cultures with the three UG stromal clones. After contact or non-contact co-culture of genetically marked embryonic cells with the stromal lines, cells were transplanted into irradiated adult recipients and tested for HSC repopulation 4 months post-transplantation. Also, 1 ee of these cells was injected directly into irradiated recipients as a control for input HSCs in the co-cultures. In the long-term in vivo repopulation assay, we found that all three stromal lines behaved similarly in their support of HSCs. As shown in Table 1, 4/9, 5/9, and 2/5 recipients were high level, multilineage repopulated with a limiting dilution (0.3 ee) of Ao cells co-cultured with 1B6, 3D4, and 3B5 respectively. Thus, these contact co-cultures support a 3-fold increase in Ao HSCs as compared to input HSC number. Moreover, despite injection of 1 ee of cultured Ao cells, no Ao HSCs were found in non-contact stromal co-cultures (0/4, 0/3, and 0/5 recipients repopulated/transplanted for 1B6, 3D4, and 3B5, respectively, or 0/12 when data are pooled). Hence, E11 Ao-derived HSCs require SC contact for maintenance and expansion ex vivo.
Table 1. Support of HSCs from different embryonic sources in contact and non-contact cultures with midgestation UG stromal clones
To directly compare support of HSCs from other E11 hematopoietic tissues to Ao HSCs, and to determine if SC contact is required for the support of these HSCs, contact and non-contact co-cultures of 1B6, 3D4 and 3B5 were established with UG, YS and FL cells. Injection of a limiting dilution (0.3 ee) of UG cells from contact co-cultures with 3D4 revealed the presence of UG HSCs in one out of six recipients, while injection of 1 ee of uncultured UG cells gave no repopulation in five recipients. This increase in UG HSC activity requires stromal contact, since no UG HSCs were found after 1 ee of such cells was transplanted. In contrast to the increase found for Ao and UG HSCs in contact co-cultures, no recipients were found repopulated after injection of 0.3 ee of contact co-cultured YS cells. YS HSC activity was also absent in the non-contact cultures as determined by transplantation of 1 ee of cells. In studies with FL cells, support of HSCs was found in contact cultures but not in non-contact cultures (data not shown). Hence, UG SC lines expand and maintain Ao and UG HSCs, but do not support YS HSCs. These results suggest that HSCs and SCs are most functionally compatible when derived from closely related embryonic tissue. Moreover, contact with UG stromal clones is essential for the support/expansion of these midgestation HSCs.
DISCUSSION
We thank all laboratory members for their helpful comments and assistance with various aspects of this work. We especially thank Robert Oostendorp, Rob Ploemacher, Trui Visser, Kam-Wing Ling, Katrin Ottersbach, Marian Peeters and Karim Hussein and the Erasmus Dierexperimenteel Centrum for animal care. We appreciate the critical review of the manuscript by Dr. Catherine Robin. This work was supported by the NIH RO DK51077 and ErasmusMC Breedtestrategie Program.
REFERENCES
Lemischka IR, Raulet DH, Mulligan RC et al. Developmental potential and dynamic behavior of hematopoietic stem cells. Cell 1986;45:917–927.
Dexter TM, Testa NG. Differentiation and proliferation of hemopoietic cells in culture. Methods Cell Biol 1976;14:387–405.
Young PE, Baumhueter S, Lasky LA et al. The sialomucin CD34 is expressed on hematopoietic cells and blood vessels during murine development. Blood 1995;85:96–105.
Tavian M, Coulombel L, Luton D et al. Aorta-associated CD34+ hematopoietic cells in the early human embryo. Blood 1996;87:67–72.
Roy V, Verfaillie CM. Soluble factor(s) produced by adult bone marrow stroma inhibit in vitro proliferation and differentiation of fetal liver BFU-E by inducing apoptosis. J Clin Invest 1997;100:912–920.
Moore KA, Ema H, Lemischka IR et al. In vitro maintenance of highly purified, transplantable hematopoietic stem cells. Blood 1997;89:4337–4347.
Coulombel L, Eaves AC, Eaves CJ et al. Enzymatic treatment of long-term human marrow cultures reveals the preferential location of primitive hemopoietic progenitors in the adherent layer. Blood 1983;62:291–297.
Breems DA, Blokland EA, Siebel KE et al. Stroma-contact prevents loss of hematopoietic stem cell quality during ex vivo expansion of CD34+ mobilized peripheral blood stem cells. Blood 1998;91:111–117.
Dzierzak E. Hematopoietic stem cells and their precursors: developmental diversity and lineage relationships. Immunol Rev 2002;187:126–138.
Muller AM, Medvinsky A, Strouboulis J et al. Development of hematopoietic stem cell activity in the mouse embryo. Immunity 1994;1:291–301.
Medvinsky A, Dzierzak E. Definitive hematopoiesis is autonomously initiated by the AGM region. Cell 1996;86:897–906.
Ohkawara JI, Ikebuchi K, Fujihara M et al. Culture system for extensive production of CD19+IgM+ cells by human cord blood CD34+ progenitors. Leukemia 1998;12:764–771.
Ohneda O, Fennie C, Zheng Z et al. Hematopoietic stem cell maintenance and differentiation are supported by embryonic aorta-gonad-mesonephros region-derived endothelium. Blood 1998;92:908–919.
Xu MJ, Tsuji K, Ueda T et al. Stimulation of mouse and human primitive hematopoiesis by murine embryonic aorta-gonad-mesonephros-derived stromal cell lines. Blood 1998;92:2032–2040.
Oostendorp RA, Harvey KN, Kusadasi N et al. Stromal cell lines from mouse aorta-gonads-mesonephros subregions are potent supporters of hematopoietic stem cell activity. Blood 2002;99:1183–1189.
Oostendorp RA, Medinsky AJ, Kusadasi N et al. Embryonal subregion-derived stromal cell lines from novel temperature-sensitive SV40 T antigen transgenic mice support hematopoiesis. J Cell Sci 2002;115:2099–2108.
Kusadasi N, Oostendorp RA, Koevoet WJ et al. Stromal cells from murine embryonic aorta-gonad-mesonephros region, liver and gut mesentery expand human umbilical cord blood-derived CAFC(week 6) in extended long-term cultures. Leukemia 2002;16:1782–1790.
Bennaceur-Griscelli A, Tourino C, Izac B et al. Murine stromal cells counteract the loss of long-term culture-initiating cell potential induced by cytokines in CD34(+)CD38(low/neg) human bone marrow cells. Blood 1999;94:529–538.
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