- Email: [email protected]
Simple Approach to Increase Donor Hematopoietic Stem Cell Dose and Improve Engraftment in the Murine Model of Allogeneic In Utero Hematopoietic Cell Transplantation
ARTICLE IN PRESS Biol Blood Marrow Transplant && (2019) &&& &&&
Biology of Blood and Marrow Transplantation journal homepage: www.bbmt.org 1
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Simple Approach to Increase Donor Hematopoietic Stem Cell Dose and Improve Engraftment in the Murine Model of Allogeneic In Utero Hematopoietic Cell Transplantation X X
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1X XJesse XD D. VrecenakD112X X , D13X XEmily A. PartridgeD214X X , D15X XErik G. PearsonD216X X , D17X XAlan W. FlakeD18X2,X *
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72 Article history: Received 30 May 2019 Accepted 22 August 2019 Keywords: In utero hematopoietic cell transplantation Hematopoietic stem cell enrichment Lineage depletion
A B S T R A C T The rationale for in utero hematopoietic cell transplantation (IUHCT) rests D19X X D20X X exploitation of normal events during on hematopoietic and immunologic ontogeny to allow allogeneic hematopoietic engraftment without myeloablative conditioning. Host hematopoietic competition is among the primary barriers to engraftment in IUHCT. In the murine modelD21X tX his can be partially overcome by delivery of larger donor cell doses, but volume is limiting. Enrichment of donor hematopoietic stem cells (HSCs) would D2X X seem to offer a more efficient approach, but such enriched populations have engrafted poorly in existing models of IUHCT. To increase HSC dose while maintaining the presence of accessory cells, we used D23X X a less stringent enrichment protocol of single-Dstep 24X X lineage depleted cells alone (Lin-) or in combination with whole donor bone marrow mononuclear cells. Our results confirm that increasing doses of HSCs in combination with bone marrowD25X X accessory cells can dramatically improve engraftment after IUHCT. This represents a practical and clinically applicable strategy to maximize the engraftment potential of the donor graft without risk of treatment-associated toxicity. © 2019 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.
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INTRODUCTION The primary rationale for in utero hematopoietic cell transplantation (IUHCT) is the exploitation of normal immunologic development to achieve donor-Dspecific 26X X tolerance for D27X X allogeneic cells. However, until recentlyD28X X most clinical and experimental efforts at IUHCT resulted in minimal or no engraftment and inconsistent donor-specific toleranceD.29X X Studies in the murine model have begun to elucidate the mechanisms of IUHCTD30X X associated donor-specific tolerance D31X X[1,2]D32X Xand have defined the potential importance of the maternal immune system [3,4] as a barrier to engraftment. In our murine model of allogeneic IUHCT, we have previously demonstrated that in the absence of maternal immune response, engraftment and tolerance are uniformly achieved by a combination of central thymic deletion and peripheral T-regulatory suppression of alloreactive lymphocytes [3]. However, even in the absence of an immune barrier, achieving therapeutically relevant levels of engraftment remains a challenge.
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Financial disclosure: See Acknowledgments on page XXXX. * Correspondence and reprint requests: Alan W. Flake, MD, Department of Surgery, Children’s Hospital of Philadelphia, Center for Fetal Research, Abramson Research Building, Room 1116B, 3615 Civic Center Boulevard, Philadelphia, PA 19104. E-mail address: fl[email protected] (A.W. Flake).
The most likely remaining barrier to engraftment is competition from the nonDmyeloablated 3X X host hematopoietic system [5]. It has been well documented that there is an excess of circulating hematopoietic stem cells (HSCs) during fetal life and that fetal HSCs are more competitive than adult HSCs in competitive repopulation assays [6-10]. An obvious strategy to improve engraftment is the use of highly enriched HSCs to increase donor HSC dose. However, our previous attempts to use highly enriched HSCs have resulted in little or no engraftment, even in congenic strain combinations (unpublished data). We reasoned that the rigorous enrichment of HSCs may negatively impact their engraftment in the fetusD34X X or that other components of the donor graft may be required to facilitate engraftment of enriched HSCs. In this study we assess the efficacy of a clinically applicable, less rigorous enrichment protocol with or without the addition of non-enriched bone marrow (BM) cells to improve engraftment.
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METHODS Mice Mice were derived from breeding colonies maintained by our laboratory. BALB/c (H2Kd+) mice were used for time-dated mating, and pregnant females underwent IUHCT at E14. All donor BM was harvested from 6- to 12-Dweek35X X old MHC-H2Kb+GFP+C57BL/6TgN(act-EGFP)OsbY01 mice (B6GFP) (originally provided by M. Okabe, Genome Information Research Center, Osaka University, Osaka, Japan). All experimental protocols were approved by the Institutional Animal Care and Use Committee at The Children’s Hospital of
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In Utero Transplantation Donor low-density mononuclear cells (MNCs) were separated by Ficoll gradient centrifugation and resuspended in PBS. For enriched transplants, lineage depleted (lin X -) X cells were isolated using a MACS lineage depletion kit (Miltenyi Biotec, Auburn, CA). For KSL X X transplantationD36X X the lineage depletion was further enriched using CD117(c-kit) APC (eBioscience, San Diego, CA), Sca-1 PE-Cy7 (BD Pharmingen, San Jose, CA), and biotinylated lineage antibody cocktail (Miltenyi BiotecD), 37X X followed by streptavidin APCCy7 (eBioscienceD)D38X X 39 X as a secondary stain. KSL cells were isolated via flow cytometric sorting using a FACSAria (BD, Franklin Lakes, NJ). At E14D40X X each fetus was injected with either 10 £ 106 BM-MNCs (WBM), 5 £ 106 lincells, 1 £ 105 KSL cells, or 10 £ 106 BM-MNCs (WBM) + 5 £ 106 lin- cells in 20 mLD41X P X BS as previously described [11]. Surviving pups were placed with a foster dam within 24 hours of birth.
population to estimate total donor HSCs administered per fetus. Our goal was to administer HSC doses in the experimental limbs that were each significantly increased relative to our control BM dose of 10 £ 106 low-density MNCs. In our control BM-MNCs the KSL fraction was determined to be .2%, D60X X giving a calculated total donor HSC content of 2 £ 104 HSCs per fetus. D61X X Within the lin- population, KSL frequency increased to 3.6%, representing an 18-fold enrichment (Figure 1). Thus, fetuses in the KSL group received 1 £ 105 HSCs, fetuses receiving 5 £ 106 lin- cells (enriched group) received 1.8 £ 105 HSCs, and fetuses receiving 10 £ 106 BM-MNCs + 5 £ 106 lin- cells (combined group) received 2 £ 105 HSCs. Lin- cells represented 1% to 3% D62X X of whole bone marrow, and MACS lineage depletion conferred >90% purity.
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Flow Cytometry Donor cells were analyzed for KSL content using CD117(c-kit) APC (eBioscienceD), 48X X Sca-1 PE-Cy7 (BD PharmingenD), 49X X and biotinylated lineage antibody cocktail (Miltenyi BiotecD), 50X X followed by streptavidin APC-Cy7 (eBioscienceD)D51X X 52 Xas a secondary stain. For chimerism analysis, peripheral blood mononuclear cells were stained with CD45 APC (BD PharmingenD). 53X X For multilineage analysis, cells were stained with CD3 PE (BD PharmingenD), 54X X B220 PE-Cy5 (BD PharmingenD), 5X X CD11b PE-Cy7 (DBD 56X X PharmingenD), 57X X and CD11c PE (BD PharmingenD). 58X X Flow cytometry was performed on a FACSCalibur (BDD). 59X X Statistics The significance of differences among groups was determined using the Student’s t-test for 2 samples assuming unequal variances. A 2-tailed P .05 was considered significant.
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Chimerism At 1, 2, 3, 4, and 8 months of ageD42X p X eripheral blood from E14-Dinjected 43X X mice was obtained via retro-orbital puncture. After mice were killed, lDiver, 4X X spleen, and BM were assessed after D45X X mechanical homogenization andD46X X passage through a 70-mM filter and RBC lysis. Donor cell chimerism was assessed as the percentage of CD45+ cells that were GFP+ by flow cytometry. Similarly, multilineage chimerism was determined based on D47X X the percentage of each cell type found to be GFP+, as described above.
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RESULTS HSC Content within Donor Cells To compare donor cell populations for HSC content, we determined the cKit+Sca-1+Lin- (KSL) frequency within each
Enrichment of the Donor Cell Graft Increases Allogeneic Engraftment AltDhough 63X X KSL cells alone failed to engraft, mean levels of peripheral blood chimerism at 1 monthD64X X were significantly increased in the enriched group as compared with WBM controls (27.4% versus 11.8%, D65X X P< D6X X .001). D67X X Unlike the WBM controls, in which chimerism levels continued to decrease throughout the period of analysis, the enriched group stabilized by 3D68X X months and showed no further decrease through 8D69X X months (Figure 2A).
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Addition of Whole Bone Marrow Cells Increases the Engraftment Potential of an Enriched Graft Strikingly, the KSL cells entirely failed to engraft in the absence of nonDenriched 70X X BM cells, despite a 5-fold increase in HSC content over the control group. Initial mean chimerism in the combined group was significantly higher than the lin- cells alone (50.3% versus 27.4%, D71X X P= D72X X .03). D73X X The combined group maintained stable engraftment averaging approximately 25% (Figure 2A), which was significantly higher than either of the other groups and suggests that a cell population within the BM may facilitate HSC engraftment. Other hematopoietic organs demonstrated similar relative improvements in engraftment
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Figure 1. HSC content of enriched population. KSL frequency within WBM (A) and Lin- cells (B), showing a 40-fold enrichment from .2% D1X X (WBM) to 3.6% (Lin-).
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Figure 2. Postnatal engraftment afterD2X X enriched IUHCT. Chimerism D3X X as a percent of GFP cells within the CD45 population. In peripheral blood (A)D4X X the addition of whole bone marrow to a Lin- population enriched for HSCDs5X X provided significant benefit over the enriched population aloneD6X aX nd an even further benefit over a nonDen7X X riched graft. Similar relative increases in chimerism were seen with enriched cells in other hematopoietic sites, including liver, spleen, and BM (B), and the facilitating effect of accessory cells was again observed. Error bars represent standard error of measurement with D8X X 12 mice per group.
Multilineage Engraftment afterD74X E X nriched IUHCT Multilineage engraftment was analyzed within the donor cell population in all mice when they were killedD.75X X The persistence of stable, multilineage chimerism at 8 months of age in all groups confirms HSC engraftment. The lineage analysis demonstrated balanced donor lineage populations without skewing of lineage representation (Figure 3), once again confirming the contribution of donor HSCsD76X X rather than any lineage-committed progenitor population. DISCUSSION IUHCT represents a promising strategy for the treatment of any disorder that can be diagnosed early in gestation and can be treated by postnatal HSCT. Potential target disorders include the hemoglobinopathiesD7X X and immunodeficiency syndromes, among others.
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There are 3D78X tX heoretical barriers to engraftment after IUHCT: an D79X X immune response to donor cells, “space” D80X X within the host hematopoietic niche, and competition D81X X from host hematopoiesis. WDork 82X X in our laboratory has shown that the previously recognized adaptive alloresponse limiting engraftment in our model [11] was secondary to maternal immunization and transfer of maternal antibodies via breast milk [3]. This can be avoided completely by maternal fostering, which was performed in this study, excluding immune response as a confounding factor and leaving host hematopoiesis as the remaining barrier. The simplest approach to overcoming host hematopoietic competition is to increase the dose of donor HSCs transplanted. We have previously increased donor cell dose and improved engraftment by use of the intravascular technique, which allows injection of a much greater volume of cells than our previous intraperitoneal technique. However, even with maximization of the dose of nonDenriched 83X X BM cells, mean levels of donor chimerism remain below the threshold likely to be required for cure of many hematopoietic diseases [12-15]. Enrichment of donor cell grafts represents an obvious and clinically applicable
(liver, spleen, BM) (Figure 2B). No significant differences in survival were observed between groups.
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Figure 3. Multilineage engraftment afterD9X eX nriched IUHCT. Distribution of engrafted GFP+ cells was found to be similar across all groups, suggesting similar patterns of HSC expansion. No significant differences were seen in chimerism for any lineage, providing evidence that the increase in chimerism results from HSC engraftment rather than any lineage-committed progenitor population. Error bars represent standard error of measurement with D10X X 12 mice per group.
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approach to increase donor HSC dose. However, all our D84X X previous attempts to engraft highly enriched HSCs by IUHCT have been disappointing with little or no engraftment achieved in allogeneic systems and very minimal engraftment observed in congenic systems. The reason for failure of engraftment of highly enriched allogeneic cells after IUHCT is speculative but includes a detrimental effect on homing or engraftment caused by the enrichment methodD85X X or D86Xother X cells in the BM that may be required D87X X to “facilitate” engraftment in the fetus. These results confirm our prior findingsD8X X and are supportive of either or both of these reasons. The use of a single lineage depletion step allowed a 9-fold increase D89X X in donor HSC dose (despite D90X X transplanting half the number of total cells) and resulted in a more than doubling of mean donor cell chimerism at 1 month of age and a sustained 5-Dfold 91X X increase at 8 months of age. This is particularly striking when compared with the absolute lack of engraftment with a similar number of highly enriched KSL cells. Thus, a much less stringent enrichment protocol appeared to markedly improve engraftment. The combination of whole BMMNCs (containing a negligible additional number of HSCs but a much higher number of cells) resulted in an additional major increase in early and late chimerism, supporting a facilitating effect of some component of the BM-MNCs on HSC engraftment. Engraftment remained twice that of the enriched group long after the anticipated contribution of short-term D92X X repopulating activity, demonstrating that their increased chimerism is the result of an increase in the absolute number of engrafted HSCDs93X X despite an equivalent number within the donor inoculum. D94X X The mechanism of the facilitating effect requires further study. Results in large animal models show that highly enriched HSCs may have engraftment defects that are reversible on D95X X addition of even low doses of T cells [16-18]. Likewise, stromal cell progenitors [19,20] and dendritic cell precursors [21] may play some role. Alternatively, the co-transplantation with BM cells may provide growth factors or paracrine effects that enhance donor cell homing, engraftment, or subsequent competition. AlthoughD96X tX his would be a likely explanation for a short-Dterm 97X X increase in donor chimerism, it is a less likely to explain an increase in long-Dterm 98X X engraftment [22]. This study is best viewed as a proof in principal that enrichment strategies can potentially improve engraftment after IUHCTD9X X and that if delivered in very high doses, adult HSCs can effectively compete in the fetal hematopoietic environment. This is a particularly attractive strategy because it requires no pharmacologic manipulation of the mother, fetus, or donor cells. It also negates the need for use of more competitive but more controversial sources of HSCs such as fetal HSCs or embryonic stem cells. Further studies are needed to determine the optimal enrichment strategy for clinical application.
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ACKNOWLEDGMENTS X X Financial disclosure: The authors have nothing to disclose. Conflict of interest statement: There are no conflicts of interest to report.
REFERENCES
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1. Kim HB, Shaaban AF, Milner R, Fichter C, Flake AW. In utero bone marrow transplantation induces donor-specific tolerance by a combination of clonal deletion and clonal anergy. J Pediatr Surg. 1999;34:726–729. discussion 729-730. 2. Nijagal A, Derderian C, Le T, et al. Direct and indirect antigen presentation lead to deletion of donor-specific T cells after in utero hematopoietic cell transplantation in mice. Blood. 2013;121:4595–4602. 3. Merianos DJ, Tiblad E, Santore MT, et al. Maternal alloantibodies induce a postnatal immune response that limits engraftment following in utero hematopoietic cell transplantation in mice. J Clin Invest. 2009;119:2590–2600. 4. Nijagal A, Wegorzewska M, Jarvis E, Le T, Tang Q, MacKenzie TC. Maternal T cells limit engraftment after in utero hematopoietic cell transplantation in mice. J Clin Invest. 2011;121:582–592. 5. Flake AW, Zanjani ED. In utero hematopoietic stem cell transplantation: ontogenic opportunities and biologic barriers. Blood. 1999;94:2179–2191. 6. Harrison DE, Astle CM. Short- and long-term multilineage repopulating hematopoietic stem cells in late fetal and newborn mice: models for human umbilical cord blood. Blood. 1997;90:174–181. 7. Harrison DE, Zhong RK, Jordan CT, Lemischka IR, Astle CM. Relative to adult marrow, fetal liver repopulates nearly five times more effectively long-term than short-term. Exp Hematol. 1997;25:293–297. 8. Jordan CT, Astle CM, Zawadzki J, Mackarehtschian K, Lemischka IR, Harrison DE. Long-term repopulating abilities of enriched fetal liver stem cells measured by competitive repopulation. Exp Hematol. 1995;23:1011–1015. 9. Leung W, Ramirez M, Civin CI. Quantity and quality of engrafting cells in cord blood and autologous mobilized peripheral blood. Biol Blood Marrow Transplant. 1999;5:69–76. 10. Rosler ES, Brandt JE, Chute J, Hoffman R. An in vivo competitive repopulation assay for various sources of human hematopoietic stem cells. Blood. 2000;96:3414–3421. 11. Peranteau WH, Endo M, Adibe OO, Flake AW. Evidence for an immune barrier after in utero hematopoietic-cell transplantation. Blood. 2007;109:1331–1333. 12. Bjorgvinsdottir H, Ding C, Pech N, Gifford MA, Li LL, Dinauer MC. Retroviral-mediated gene transfer of gp91phox into bone marrow cells rescues defect in host defense against Aspergillus fumigatus in murine X-linked chronic granulomatous disease. Blood. 1997;89:41–48. 13. Bauer Jr. TR, Creevy KE, Gu YC, et al. Very low levels of donor CD18+ neutrophils following allogeneic hematopoietic stem cell transplantation reverse the disease phenotype in canine leukocyte adhesion deficiency. Blood. 2004;103:3582–3589. 14. Andreani M, Nesci S, Lucarelli G, et al. Long-term survival of ex-thalassemic patients with persistent mixed chimerism after bone marrow transplantation. Bone Marrow Transplant. 2000;25:401–404. 15. Walters MC, Patience M, Leisenring W, et al. Bone marrow transplantation for sickle cell disease. N Engl J Med. 1996;335:369–376. 16. Shields LE, Gaur LK, Gough M, Potter J, Sieverkropp A, Andrews RG. In utero hematopoietic stem cell transplantation in nonhuman primates: the role of T cells. Stem Cells. 2003;21:304–314. 17. Crombleholme TM, Harrison MR, Zanjani ED. In utero transplantation of hematopoietic stem cells in sheep: the role of T cells in engraftment and graft-versus-host disease. J Pediatr Surg. 1990;25:885–892. 18. Petersen SM, Gendelman M, Murphy KM, et al. Use of T-cell antibodies for donor dosaging in a canine model of in utero hematopoietic stem cell transplantation. Fetal Diagn Ther. 2007;22:175–179. 19. Almeida-Porada G, Flake AW, Glimp HA, Zanjani ED. Cotransplantation of stroma results in enhancement of engraftment and early expression of donor hematopoietic stem cells in utero. Exp Hematol. 1999;27:1569–1575. 20. Almeida-Porada G, Porada CD, Tran N, Zanjani ED. Cotransplantation of human stromal cell progenitors into preimmune fetal sheep results in early appearance of human donor cells in circulation and boosts cell levels in bone marrow at later time points after transplantation. Blood. 2000;95:3620–3627. 21. Huang Y, Bozulic LD, Miller T, Xu H, Hussain LR, Ildstad ST. CD8α+ plasmacytoid precursor DCs induce antigen-specific regulatory T cells that enhance HSC engraftment in vivo. Blood. 2011;117:2494–2505. 22. Shaaban AF, Kim HB, Gaur L, Liechty KW, Flake AW. Prenatal transplantation of cytokine-stimulated marrow improves early chimerism in a resistant strain combination but results in poor long-term engraftment. Exp Hematol. 2006;34:1278–1287.
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Simple Approach to Increase Donor Hematopoietic Stem Cell Dose and Improve Engraftment in the Murine Model of Allogeneic In Utero Hematopoietic Cell Transplantation X X
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1X XJesse XD D. VrecenakD112X X , D13X XEmily A. PartridgeD214X X , D15X XErik G. PearsonD216X X , D17X XAlan W. FlakeD18X2,X *
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72 Article history: Received 30 May 2019 Accepted 22 August 2019 Keywords: In utero hematopoietic cell transplantation Hematopoietic stem cell enrichment Lineage depletion
A B S T R A C T The rationale for in utero hematopoietic cell transplantation (IUHCT) rests D19X X D20X X exploitation of normal events during on hematopoietic and immunologic ontogeny to allow allogeneic hematopoietic engraftment without myeloablative conditioning. Host hematopoietic competition is among the primary barriers to engraftment in IUHCT. In the murine modelD21X tX his can be partially overcome by delivery of larger donor cell doses, but volume is limiting. Enrichment of donor hematopoietic stem cells (HSCs) would D2X X seem to offer a more efficient approach, but such enriched populations have engrafted poorly in existing models of IUHCT. To increase HSC dose while maintaining the presence of accessory cells, we used D23X X a less stringent enrichment protocol of single-Dstep 24X X lineage depleted cells alone (Lin-) or in combination with whole donor bone marrow mononuclear cells. Our results confirm that increasing doses of HSCs in combination with bone marrowD25X X accessory cells can dramatically improve engraftment after IUHCT. This represents a practical and clinically applicable strategy to maximize the engraftment potential of the donor graft without risk of treatment-associated toxicity. © 2019 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.
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INTRODUCTION The primary rationale for in utero hematopoietic cell transplantation (IUHCT) is the exploitation of normal immunologic development to achieve donor-Dspecific 26X X tolerance for D27X X allogeneic cells. However, until recentlyD28X X most clinical and experimental efforts at IUHCT resulted in minimal or no engraftment and inconsistent donor-specific toleranceD.29X X Studies in the murine model have begun to elucidate the mechanisms of IUHCTD30X X associated donor-specific tolerance D31X X[1,2]D32X Xand have defined the potential importance of the maternal immune system [3,4] as a barrier to engraftment. In our murine model of allogeneic IUHCT, we have previously demonstrated that in the absence of maternal immune response, engraftment and tolerance are uniformly achieved by a combination of central thymic deletion and peripheral T-regulatory suppression of alloreactive lymphocytes [3]. However, even in the absence of an immune barrier, achieving therapeutically relevant levels of engraftment remains a challenge.
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Financial disclosure: See Acknowledgments on page XXXX. * Correspondence and reprint requests: Alan W. Flake, MD, Department of Surgery, Children’s Hospital of Philadelphia, Center for Fetal Research, Abramson Research Building, Room 1116B, 3615 Civic Center Boulevard, Philadelphia, PA 19104. E-mail address: fl[email protected] (A.W. Flake).
The most likely remaining barrier to engraftment is competition from the nonDmyeloablated 3X X host hematopoietic system [5]. It has been well documented that there is an excess of circulating hematopoietic stem cells (HSCs) during fetal life and that fetal HSCs are more competitive than adult HSCs in competitive repopulation assays [6-10]. An obvious strategy to improve engraftment is the use of highly enriched HSCs to increase donor HSC dose. However, our previous attempts to use highly enriched HSCs have resulted in little or no engraftment, even in congenic strain combinations (unpublished data). We reasoned that the rigorous enrichment of HSCs may negatively impact their engraftment in the fetusD34X X or that other components of the donor graft may be required to facilitate engraftment of enriched HSCs. In this study we assess the efficacy of a clinically applicable, less rigorous enrichment protocol with or without the addition of non-enriched bone marrow (BM) cells to improve engraftment.
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METHODS Mice Mice were derived from breeding colonies maintained by our laboratory. BALB/c (H2Kd+) mice were used for time-dated mating, and pregnant females underwent IUHCT at E14. All donor BM was harvested from 6- to 12-Dweek35X X old MHC-H2Kb+GFP+C57BL/6TgN(act-EGFP)OsbY01 mice (B6GFP) (originally provided by M. Okabe, Genome Information Research Center, Osaka University, Osaka, Japan). All experimental protocols were approved by the Institutional Animal Care and Use Committee at The Children’s Hospital of
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Philadelphia and followed guidelines set forth in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
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In Utero Transplantation Donor low-density mononuclear cells (MNCs) were separated by Ficoll gradient centrifugation and resuspended in PBS. For enriched transplants, lineage depleted (lin X -) X cells were isolated using a MACS lineage depletion kit (Miltenyi Biotec, Auburn, CA). For KSL X X transplantationD36X X the lineage depletion was further enriched using CD117(c-kit) APC (eBioscience, San Diego, CA), Sca-1 PE-Cy7 (BD Pharmingen, San Jose, CA), and biotinylated lineage antibody cocktail (Miltenyi BiotecD), 37X X followed by streptavidin APCCy7 (eBioscienceD)D38X X 39 X as a secondary stain. KSL cells were isolated via flow cytometric sorting using a FACSAria (BD, Franklin Lakes, NJ). At E14D40X X each fetus was injected with either 10 £ 106 BM-MNCs (WBM), 5 £ 106 lincells, 1 £ 105 KSL cells, or 10 £ 106 BM-MNCs (WBM) + 5 £ 106 lin- cells in 20 mLD41X P X BS as previously described [11]. Surviving pups were placed with a foster dam within 24 hours of birth.
population to estimate total donor HSCs administered per fetus. Our goal was to administer HSC doses in the experimental limbs that were each significantly increased relative to our control BM dose of 10 £ 106 low-density MNCs. In our control BM-MNCs the KSL fraction was determined to be .2%, D60X X giving a calculated total donor HSC content of 2 £ 104 HSCs per fetus. D61X X Within the lin- population, KSL frequency increased to 3.6%, representing an 18-fold enrichment (Figure 1). Thus, fetuses in the KSL group received 1 £ 105 HSCs, fetuses receiving 5 £ 106 lin- cells (enriched group) received 1.8 £ 105 HSCs, and fetuses receiving 10 £ 106 BM-MNCs + 5 £ 106 lin- cells (combined group) received 2 £ 105 HSCs. Lin- cells represented 1% to 3% D62X X of whole bone marrow, and MACS lineage depletion conferred >90% purity.
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Flow Cytometry Donor cells were analyzed for KSL content using CD117(c-kit) APC (eBioscienceD), 48X X Sca-1 PE-Cy7 (BD PharmingenD), 49X X and biotinylated lineage antibody cocktail (Miltenyi BiotecD), 50X X followed by streptavidin APC-Cy7 (eBioscienceD)D51X X 52 Xas a secondary stain. For chimerism analysis, peripheral blood mononuclear cells were stained with CD45 APC (BD PharmingenD). 53X X For multilineage analysis, cells were stained with CD3 PE (BD PharmingenD), 54X X B220 PE-Cy5 (BD PharmingenD), 5X X CD11b PE-Cy7 (DBD 56X X PharmingenD), 57X X and CD11c PE (BD PharmingenD). 58X X Flow cytometry was performed on a FACSCalibur (BDD). 59X X Statistics The significance of differences among groups was determined using the Student’s t-test for 2 samples assuming unequal variances. A 2-tailed P .05 was considered significant.
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Chimerism At 1, 2, 3, 4, and 8 months of ageD42X p X eripheral blood from E14-Dinjected 43X X mice was obtained via retro-orbital puncture. After mice were killed, lDiver, 4X X spleen, and BM were assessed after D45X X mechanical homogenization andD46X X passage through a 70-mM filter and RBC lysis. Donor cell chimerism was assessed as the percentage of CD45+ cells that were GFP+ by flow cytometry. Similarly, multilineage chimerism was determined based on D47X X the percentage of each cell type found to be GFP+, as described above.
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RESULTS HSC Content within Donor Cells To compare donor cell populations for HSC content, we determined the cKit+Sca-1+Lin- (KSL) frequency within each
Enrichment of the Donor Cell Graft Increases Allogeneic Engraftment AltDhough 63X X KSL cells alone failed to engraft, mean levels of peripheral blood chimerism at 1 monthD64X X were significantly increased in the enriched group as compared with WBM controls (27.4% versus 11.8%, D65X X P< D6X X .001). D67X X Unlike the WBM controls, in which chimerism levels continued to decrease throughout the period of analysis, the enriched group stabilized by 3D68X X months and showed no further decrease through 8D69X X months (Figure 2A).
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Addition of Whole Bone Marrow Cells Increases the Engraftment Potential of an Enriched Graft Strikingly, the KSL cells entirely failed to engraft in the absence of nonDenriched 70X X BM cells, despite a 5-fold increase in HSC content over the control group. Initial mean chimerism in the combined group was significantly higher than the lin- cells alone (50.3% versus 27.4%, D71X X P= D72X X .03). D73X X The combined group maintained stable engraftment averaging approximately 25% (Figure 2A), which was significantly higher than either of the other groups and suggests that a cell population within the BM may facilitate HSC engraftment. Other hematopoietic organs demonstrated similar relative improvements in engraftment
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Figure 1. HSC content of enriched population. KSL frequency within WBM (A) and Lin- cells (B), showing a 40-fold enrichment from .2% D1X X (WBM) to 3.6% (Lin-).
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Figure 2. Postnatal engraftment afterD2X X enriched IUHCT. Chimerism D3X X as a percent of GFP cells within the CD45 population. In peripheral blood (A)D4X X the addition of whole bone marrow to a Lin- population enriched for HSCDs5X X provided significant benefit over the enriched population aloneD6X aX nd an even further benefit over a nonDen7X X riched graft. Similar relative increases in chimerism were seen with enriched cells in other hematopoietic sites, including liver, spleen, and BM (B), and the facilitating effect of accessory cells was again observed. Error bars represent standard error of measurement with D8X X 12 mice per group.
Multilineage Engraftment afterD74X E X nriched IUHCT Multilineage engraftment was analyzed within the donor cell population in all mice when they were killedD.75X X The persistence of stable, multilineage chimerism at 8 months of age in all groups confirms HSC engraftment. The lineage analysis demonstrated balanced donor lineage populations without skewing of lineage representation (Figure 3), once again confirming the contribution of donor HSCsD76X X rather than any lineage-committed progenitor population. DISCUSSION IUHCT represents a promising strategy for the treatment of any disorder that can be diagnosed early in gestation and can be treated by postnatal HSCT. Potential target disorders include the hemoglobinopathiesD7X X and immunodeficiency syndromes, among others.
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There are 3D78X tX heoretical barriers to engraftment after IUHCT: an D79X X immune response to donor cells, “space” D80X X within the host hematopoietic niche, and competition D81X X from host hematopoiesis. WDork 82X X in our laboratory has shown that the previously recognized adaptive alloresponse limiting engraftment in our model [11] was secondary to maternal immunization and transfer of maternal antibodies via breast milk [3]. This can be avoided completely by maternal fostering, which was performed in this study, excluding immune response as a confounding factor and leaving host hematopoiesis as the remaining barrier. The simplest approach to overcoming host hematopoietic competition is to increase the dose of donor HSCs transplanted. We have previously increased donor cell dose and improved engraftment by use of the intravascular technique, which allows injection of a much greater volume of cells than our previous intraperitoneal technique. However, even with maximization of the dose of nonDenriched 83X X BM cells, mean levels of donor chimerism remain below the threshold likely to be required for cure of many hematopoietic diseases [12-15]. Enrichment of donor cell grafts represents an obvious and clinically applicable
(liver, spleen, BM) (Figure 2B). No significant differences in survival were observed between groups.
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Figure 3. Multilineage engraftment afterD9X eX nriched IUHCT. Distribution of engrafted GFP+ cells was found to be similar across all groups, suggesting similar patterns of HSC expansion. No significant differences were seen in chimerism for any lineage, providing evidence that the increase in chimerism results from HSC engraftment rather than any lineage-committed progenitor population. Error bars represent standard error of measurement with D10X X 12 mice per group.
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approach to increase donor HSC dose. However, all our D84X X previous attempts to engraft highly enriched HSCs by IUHCT have been disappointing with little or no engraftment achieved in allogeneic systems and very minimal engraftment observed in congenic systems. The reason for failure of engraftment of highly enriched allogeneic cells after IUHCT is speculative but includes a detrimental effect on homing or engraftment caused by the enrichment methodD85X X or D86Xother X cells in the BM that may be required D87X X to “facilitate” engraftment in the fetus. These results confirm our prior findingsD8X X and are supportive of either or both of these reasons. The use of a single lineage depletion step allowed a 9-fold increase D89X X in donor HSC dose (despite D90X X transplanting half the number of total cells) and resulted in a more than doubling of mean donor cell chimerism at 1 month of age and a sustained 5-Dfold 91X X increase at 8 months of age. This is particularly striking when compared with the absolute lack of engraftment with a similar number of highly enriched KSL cells. Thus, a much less stringent enrichment protocol appeared to markedly improve engraftment. The combination of whole BMMNCs (containing a negligible additional number of HSCs but a much higher number of cells) resulted in an additional major increase in early and late chimerism, supporting a facilitating effect of some component of the BM-MNCs on HSC engraftment. Engraftment remained twice that of the enriched group long after the anticipated contribution of short-term D92X X repopulating activity, demonstrating that their increased chimerism is the result of an increase in the absolute number of engrafted HSCDs93X X despite an equivalent number within the donor inoculum. D94X X The mechanism of the facilitating effect requires further study. Results in large animal models show that highly enriched HSCs may have engraftment defects that are reversible on D95X X addition of even low doses of T cells [16-18]. Likewise, stromal cell progenitors [19,20] and dendritic cell precursors [21] may play some role. Alternatively, the co-transplantation with BM cells may provide growth factors or paracrine effects that enhance donor cell homing, engraftment, or subsequent competition. AlthoughD96X tX his would be a likely explanation for a short-Dterm 97X X increase in donor chimerism, it is a less likely to explain an increase in long-Dterm 98X X engraftment [22]. This study is best viewed as a proof in principal that enrichment strategies can potentially improve engraftment after IUHCTD9X X and that if delivered in very high doses, adult HSCs can effectively compete in the fetal hematopoietic environment. This is a particularly attractive strategy because it requires no pharmacologic manipulation of the mother, fetus, or donor cells. It also negates the need for use of more competitive but more controversial sources of HSCs such as fetal HSCs or embryonic stem cells. Further studies are needed to determine the optimal enrichment strategy for clinical application.
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ACKNOWLEDGMENTS X X Financial disclosure: The authors have nothing to disclose. Conflict of interest statement: There are no conflicts of interest to report.
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