Hematopoietic stem cell transplantation without irradiation

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1 nature methods Hematopoietic stem cell transplantation without irradiation Claudia Waskow, Vikas Madan, Susanne Bartels, Céline Costa, Rosel Blasig & Hans-Reimer Rodewald Supplementary figures and text: Supplementary Figure 1 Reconstitution of non-irradiated Rag -/- -/- W/Wv γ c Kit mice by donor HSC for at least 16 weeks (long-term reconstitution) Supplementary Figure 2 HSC reconstitution of non-irradiated Rag -/- -/- W/Wv γ c Kit and irradiated C57BL/6 wild type mice Supplementary Figure 3 Cell-intrinsic defects in HSC maintenance in Bclaf1- deficient mice Supplementary Figure 4 Reduced absolute numbers of long-term HSC in Rag -/- -/- γ c Kit W/Wv mice Supplementary Table 1 Stem cell failure of Bclaf1 -/- HSC revealed in Rag -/- γ c -/- Supplementary Results Supplementary Discussion Supplementary Methods Kit W/Wv recipients

2 Supplementary Figure 1 legend Reconstitution of non-irradiated Rag2 -/- γ -/- c Kit W/Wv mice by donor HSCs for at least 16 weeks (long-term reconstitution). 1.5 x 10 5 purified Lin - cells from 129/Ola x C57BL/6 mice (see text) were injected into non-irradiated Rag2 -/- γ -/- c Kit W/Wv mice. 16 weeks after transfer, donor cells were quantified by flow cytometry using high Kit expression as donor marker. (a) The fraction of Kit + cells per Lin - cells was comparable in C57BL/6 (mean = 21.6 ± 6.4% [one standard deviation]), and in transplanted Rag2 -/- γ -/- c Kit W/Wv (19.8 ± 6.1%) (P = 0.32 comparing C57BL/6 versus transplanted Rag2 -/- γ -/- c Kit W/Wv; not significant). Kit + cells were undetectable in non-transplanted Rag2 -/- γ -/- c Kit W/Wv mice. (b). The fraction of Kit + Sca-1 + cells per Lin - cells was similar in C57BL/6 (2.6 ± 1.7%), and in transplanted Rag2 -/- γ -/- c Kit W/Wv (2.1 ± 1.5%) (P = 0.37 comparing C57BL/6 versus transplanted Rag2 -/- γ -/- c Kit W/Wv ; not significant). Kit + Sca-1 + cells were absent from non-transplanted Rag2 -/- γ -/- c Kit W/Wv mice.

3 Supplementary Figure 2 legend HSC reconstitution of non-irradiated Rag2 -/- γ -/- c Kit W/Wv and irradiated C57BL/6 wildtype mice. (a) Total bone marrow cells from B6.SJL (CD ) mice were injected into lethally irradiated wild-type C57BL/6 (CD ) mice (closed squares), or non-irradiated Rag2 -/- γ -/- c Kit W/Wv (CD ) mice (open squares), and analyzed after 7 weeks. The graph shows percentages of the donor cell contribution to Lin - bone marrow cells as a function of the injected number of total bone marrow cells as indicated in the figure. *10 4 congenic B6.SJL bone marrow cells did not protect lethally irradiated wild-type recipients precluding analysis of donor contribution. Each symbol represents an individual mouse, and the bar indicates the mean for each experimental group. (b) Red blood cell parameters in the peripheral blood of reconstituted Rag2 -/- γ -/- c Kit W/Wv mice were improved in a donor cell dose-dependent manner at 7 weeks after bone marrow transfer. Graphs show mean cellular volume (MCV) and red blood cell numbers (RBC) in wild-type (wt) mice, and in non-injected Rag2 -/- γ -/- c Kit W/Wv mice and in Rag2 -/- γ -/- c Kit W/Wv mice injected with the indicated number of total B6.SJL bone marrow cells.

4 Each symbol represents an individual mouse, and the bar indicates the mean for each experimental group.

5 Supplementary Figure 3 legend Cell-intrinsic defects in HSC maintenance in Bclaf1-deficient mice. (a) The genotype of Bclaf1-deficient mice was analyzed by Southern blot (Supplementary Methods). The probe detected a 10 kb fragment for the wild-type allele and a 5 kb fragment for the mutated allele. Bclaf1 -/-, Bclaf1 +/-, Bclaf1 +/+ genotypes are shown. (b) Kinetics of the increase in RBC numbers in Rag2 -/- γ -/- c Kit W/Wv mice transplanted with 10 4 Kit + Lin - bone marrow cells from wild-type (circles) or Bclaf1 -/- (squares) mice. As controls, RBC numbers were determined in wild-type mice (inversed triangles) and non-transplanted Rag2 -/- γ -/- c Kit W/Wv mice (triangles). The symbols represent the mean ± one standard deviation of two to five analyzed mice per time point and genotype. (c) Hematopoietic progenitor cells (10 4 Kit + Lin - ) of 10 days old wild-type or Bclaf1 -/- bone marrow cells were transplanted into non-irradiated Rag2 -/- γ -/- c Kit W/Wv mice. After 4.5 months, HSC reconstitution of primary recipients was analyzed (1 st transfer). At this time point, 1000 Kit + Sca-1 + Lin - cells were sorted from the primary recipients, and were transferred into secondary recipients. After 3 months, HSC reconstitution was analyzed in secondary recipients (2 nd transfer). Dot plots show expression of Kit and Sca-1 on Lin - bone marrow cells. Percentages of Kit + Sca-1 + Lin - cells are indicated next to the gate, and absolute numbers of Kit + Sca-1 + Lin - cells, given within each plot, refer to the mean ± one standard deviation for the number (n) of mice analyzed.

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7 Supplementary Figure 4 legend Reduced absolute numbers of long-term HSCs in Rag2 -/- γ -/- c Kit W/Wv mice. (a) Total bone marrow cellularity per two femura is shown for wild-type (WB-Kit +/+ ) mice, Rag2 -/- γ -/- c mice, Kit W/Wv mice, and Rag2 -/- γ -/- c Kit W/Wv mice. Each symbol represents an individual mouse, and the bar represents the mean of each experimental group. (b) Bone marrow cells, gated as Kit +/lo Sca-1 + Lineage (CD3, CD4, CD8, B220, CD19, Ter119, CD11b, Gr-1) cells were further analyzed for expression of CD34 and Flk2. The same Kit +/lo gate was used for all genotypes to accommodate stem cells from all strains, regardless of their Kit expression level. Considering total bone marrow cellularity, and according to percentages obtained by flow cytometry, absolute numbers of Kit + Sca-1 + Lin - CD34 - Flk2 - long-term-hsc-containing cells were determined for wild-type (WB-Kit +/+ ) mice, Rag2 -/- γ -/- c mice, Kit W/Wv mice, and Rag2 -/- γ -/- c Kit W/Wv mice. Each symbol represents an individual mouse, and the bars represent the mean of each experimental group. Supplementary Table 1 Stem cell failure of Bclaf1 -/- HSCs revealed in Rag2 -/- γ c -/- Kit W/Wv recipients Organ Cell type Spleen T cells Spleen B cells Thymus Pre T cells Bone marrow HSCs wt KO wt KO wt KO wt KO 1 st transfer 3/3 4/4 3/3 4/4 3/3 4/4 3/3 4/4 2 nd transfer 3/3 2/5 3/3 3/5 ND ND 3/3 0/5 Transplantation of Kit + Lin - and Kit + Sca-1 + Lin - cells was performed as described in Supplementary Fig. 3. Donor-derived mature spleen T cells (CD45 + CD3 + ), B cells (CD45 + CD19 + ), thymic pre T cells (Lin - CD25 + ), and bone marrow HSCs (Kit + Sca- 1 + Lin - ) were analyzed in primary (1 st transfer) and secondary (2 nd transfer) recipients of wild-type (wt) or Bclaf1 -/- (KO) cells. Numbers show successful reconstitution of the

8 indicated cell lineage per total number of recipients. Supplementary Results Thymocyte numbers in mice analyzed in Fig. 1a-d Consistent with strong HSC engraftment in transplanted Rag2 -/- γ -/- c Kit W/Wv mice, thymocyte numbers were comparable in two non-reconstituted wild-type mice (6.7x10 7, 3.6x10 7 ), and in two reconstituted Rag2 -/- γ -/- c Kit W/Wv (1.29x10 8, 3.7x10 7 ). In contrast, thymocyte numbers were more than 10-fold lower in two reconstituted Rag2 -/- γ -/- c mice (1.5x10 8, 5x10 5 ), indicating transient, or only marginal thymopoiesis in the absence of robust HSC engraftment in Rag2 -/- γ -/- c mice. Reconstitution efficiency of Rag2 -/- γ -/- c Kit W/Wv mice Reconstitution efficiency was assessed by comparing lethally irradiated wild-type (C57BL/6) and non-irradiated Rag2 -/- γ -/- c Kit W/Wv mice. Donor cells (B6.SJL; H-2 b ) were syngeneic for C57BL/6 cells (H-2 b ), and shared the parental H-2 b with Rag2 -/- γ -/- c Kit W/Wv mice. For each combination, donor B6.SJL (CD ) and host (CD ) differed in the CD45 allele. The contribution of donor cells to the Lin - compartment of the host was donor cell dose-dependent, and comparable between lethally irradiated wildtype and Rag2 -/- γ -/- c Kit W/Wv mice (Supplementary Fig. 2a). Engraftment following very low numbers of HSCs (10 4 total bone marrow cells) was only measurable in Rag2 -/- γ -/- c Kit W/Wv mice, but not in irradiated wild-type mice, because this low cell number failed to provide sufficient radioprotection for lethally irradiated wild-type mice (Supplementary Fig. 2a).

9 HSC cell dose-dependent cure of the macrocytic anemia in Rag2 -/- γ -/- c Kit W/Wv mice Because Rag2 -/- γ -/- c Kit W/Wv mice, as Kit W/Wv mice 1, suffer from a characteristic macrocytic anemia, we analyzed the impact of titrated numbers of donor bone marrow cells on anemic parameters. Peripheral blood (Supplementary Fig. 2b) was analyzed using an automated hemocytometer according to the manufacturer s instructions (ADVIA120; Bayer, Leverkusen, Germany). Both mean cellular volume (MCV) and red blood cell (RBC) numbers were corrected in a dose-dependent manner, and as few as 10 4 total bone marrow cells improved the anemia (Supplementary Fig. 2b). Analysis of Bclaf1 -/- HSCs in Rag2 -/- γ -/- c Kit W/Wv mice To provide a second example for a stem cell mutant analysis, HSCs from knockout mice deficient for the the Bcl2-associated transcription factor 1 (Bclaf1) gene 2 were serially transplanted into non-irradiated Rag2 -/- γ -/- c Kit W/Wv mice (Supplementary Fig. 3). Bclaf1 - /- mice were generated by homologous recombination in ES cells, and the correct targeting was verified by Southern blot hybridization (Supplementary Fig. 3a; and Blasig and Horak, manuscript in preparation). Lethality of Bclaf1 -/- mice within the first 3 weeks after birth precluded analysis of HSCs in adult Bclaf1 -/- mice. However, a 2-fold reduction of the absolute number of HSCs in 10 days old mutants (Supplementary Fig. 3c) suggested that hematopoietic failure may be an underlying cause of the premature death, possibly pointing at a role for Bclaf1 in the generation or function of HSCs. Reconstitution of the HSC compartment in primary Rag2 -/- γ -/- c Kit W/Wv recipients was monitored by the kinetics of the cure of the anemia which was comparable from wild-type or Bclaf1 -/- bone marrow (Supplementary Fig. 3b). In addition, 4.5 months after transplantation, both wild-type and Bclaf1 -/- bone marrow had successfully given rise to donor-derived T and B cells in all transplanted mice (Supplementary Table 1). Similar to HSCs in young Bclaf1 -/- mice, numbers of HSCs in primary Rag2 -/- γ -/- c Kit W/Wv mice were reduced 2-fold compared to wild-type and Bclaf1-deficient HSCs (Supplementary Fig. 3c). More dramatic defects were uncovered in secondary Rag2 -/-

10 γ -/- c Kit W/Wv recipients that were transplanted with 1000 sorted wild-type or Bclaf1 -/- Kit + Sca-1 + Lin - cells retrieved from primary Rag2 -/- γ -/- c Kit W/Wv recipients 4.5 months after the first transplantation. Analysis of the bone marrow in secondary Rag2 -/- γ -/- c Kit W/Wv recipients 3 months after transplantation still revealed HSC engraftment in the case of wild-type HSCs (Supplementary Fig. 3c, left). In contrast, HSCs, and, in fact, any Kit + cells were absent from secondary Rag2 -/- γ -/- c Kit W/Wv bone marrow after serial transplantation of Bclaf1 -/- HSCs (Supplementary Fig. 3c, right). Consistent with stem cell exhaustion between the first and second transplantation, Bclaf1 -/- HSCs failed to reconstitute the HSC compartment in all secondary recipients (Supplementary Fig. 3c and Supplementary Table 1). In keeping with this lack of sustained hematopoiesis, T and B cells were reconstituted in only 2 or 3 out of 5 secondary recipients, respectively, receiving Bclaf1 -/- HSCs (Supplementary Table 1). In contrast, wild-type HSCs successfully generated T and B cells as well as HSCs in all secondary recipient mice. We conclude that Bclaf1 -/- HSCs failed to engraft in the HSC compartment of secondary recipients, suggesting a crucial function for Bclaf1 in HSC maintenance. These findings call for a detailed characterization of the underlying defect. Supplementary Discussion Considerations on parent into F1 transplantations in Kit W/Wv recipients We state in the manuscript that non-irradiated Kit W/Wv mice, which are F1 hybrids between WB-Kit W/+ (H-2 j ) and C57Bl/6-Kit Wv/+ (H-2 b ) parents, accept syngeneic F1 (H-2 j x b ), and, partially, parental HSCs. While Kit W/Wv mice fully reject WB bone marrow, they partially accept C57BL/6 bone marrow 3. Of note, both for CFU-S and for long-term engraftment, transplantation of C57BL/6 (parental) bone marrow into Kit W/Wv mice is known to be inferior to transplantation of WBB6 (= syngeneic F1) bone marrow into Kit W/Wv mice 3. This has been attributed to F1 hybrid resistance 3, and it is, along with the rejection of allogeneic HSCs by Kit W/Wv mice 1, probably the major reason for why Kit W/Wv mice have not been routinely used for adoptive transfer analyses of HSCs.

11 Size of the endogenous HSC compartments in wild-type and mutant mice Comparison of absolute numbers of the endogenous long-term HSC-containing Kit + Sca-1 + Lin - CD34 - Flk2 - population in wild-type mice, Rag2 -/- γ -/- c mice, Kit W/Wv mice and Rag2 -/- γ -/- c Kit W/Wv mice revealed a 4.5 fold reduction comparing Rag2 -/- γ -/- c Kit W/Wv mice and wild-type mice (Supplementary Fig. 4). Based on our engraftment studies, and on CFU-S formation, we conclude that these endogenous HSCs in Rag2 -/- γ -/- c Kit W/Wv mice cannot compete in their niches against Kit + HSCs even in the absence of any exogenous myeloablation. As a result of this deficiency, and the reduced stem cell numbers, bone marrow and spleen niches of non-irradiated Rag2 -/- γ -/- c Kit W/Wv mice are freely accessible for donor stem and progenitor cells. In addition, engraftment of these niches by allogeneic HSCs is absolutely dependent on lack of Rag2 and γ c expression in Rag2 -/- γ -/- c Kit W/Wv mice. Supplementary Methods Mice DBA/2 (H-2 d ), BALB/c (H-2 d ), C57BL/6 (B6) H-2 b ), C57BL/6.SJL (B6.SJL; H-2 b ) were from Jackson Laboratory, Bar Harbour, USA, and Rag2 -/- γ -/- c were from Taconic. To generate Rag2 -/- γ -/- c Kit W/Wv mice, we used WB-Kit W/+ and C57BL/6-Kit Wv/+ which we obtained in 1994 from Japan-SLC, Shizuoka, Japan. These strains are termed WB-S/+ for WB-Kit W/+ and C57BL/6-Wv/+ for C57BL/6-Kit Wv/+ by Japan-SLC, which obtained the mice in 1981 from the Institute of Cancer Research, Osaka University School of Medicine. The parental strains of these mice were WB/ReJ-Kit W /J and C57BL/6J-Kit W-v /J from the from Jackson Laboratory 4. WBB6F1-Kit W/Wv (Kit W/Wv ) mice, and Rag2 -/- γ -/- c Kit W/Wv mice were continuously bred in our animal facility as described before 5. By way of their generation, Rag2 -/- γ -/- c Kit W/Wv mice co-express the parental haplotypes H-2 j and H-2 b.

12 Flow Cytometry Bone marrow cells were flushed from 2 femura, counted and either directly used (total bone marrow) or labeled in 100 µl PBS with 5% FCS for 40 on ice and sorted for HSC containing Kit + Sca-1 + Lineage (CD3, CD4, CD8, B220, CD19, Ter119, CD11b, Gr-1) cells. All sorts and analysis were performed using a FACS Aria, or FACS Calibur or CantoII instruments (all BD). Antibodies used were: Gr-1 (RB6-8C5), Mac-1 (M1/70.15), CD3 (145-2C11) CD4 (H129.19) CD8 (53-6.7), CD19 (1D3), CD34 (RAM34), CD45.1 (A20-1.7), CD45R (RA3.6B2), CD117 (2B8), CD135 (A2F10.1), H-2K d (SF1-1.1), Ter119 (Ter119) Sca-1 (E ) (all BD-Pharmingen). Flow cytometry and cell sorting were done as described 6. HSC transplantation and CFU-S assay C57BL/6 recipient mice were lethally irradiated (950 cgy; split dose with 2h time gap between both doses) and 2 hours later injected with cells in 200 µl PBS with 5% FCS. For Fig. 1a-d, 2000 purified Kit + Sca-1 + Lin - from BALB/c mice (H-2 d ) were injected into non-irradiated wild-type C57BL/6 (H-2 b ) mice, Rag2 -/- γ -/- c (H-2 b ) mice, Kit W/Wv (H-2 j x b ) mice and Rag2 -/- γ -/- c Kit W/Wv (H-2 j x b ) mice. In this combination, donors and recipients were hence fully MHC-mismatched. The donor contribution to bone marrow and spleen was analyzed by flow cytometry using the indicated phenotypes. In the CFU-S assay, mice were irradiated (950 cgy), or left non-irradiated as indicated, and spleens were removed 11 days later, fixed in Bouin s solution and photographed for visible colonies. In Fig. 1e-j, C57BL/6 and DBA/2 donor cells were 2x10 5 total bone marrow cells, and BALB/c donor cells were 2000 sorted Kit + Lin - cells. HSC mutant mice Mll5-deficient mice have been described 7. Bclaf1-deficient mice were generated in the FMP, Berlin, Germany (Blasig and Horak, manuscript in preparation) by homologous recombination in ES cells, and the genotype of mutant mice was analyzed by Southern blot hybridization after digestion of genomic mouse tail DNA with PstI. Blots were hybridized simultaneously with two probes generated by PCR using ES cell DNA as

13 template. Probe 1: forward primer: 5 -CCATCACTCTTGGCTTA - 3, reverse primer: 5 GCA TGC ATG GAG GCA AAT CAC 3 ; Probe 2: forward primer: 5 ACA GTC TAG ATA TGA GAA ATC AGA 3, reverse primer: 5 CCA GCA CTC TTA TTC AGA AAA CTG 3. Both fragments were labelled with 32 P-dCTP using the Amersham TM Rediprime TM II Random Primer Labelling System (GE Healthcare, Buckinghanshire, UK). Supplementary References 1. Russell, E. S. Adv. Genet 20, (1979). 2. Kasof, G. M., Goyal, L. & White, E. Mol. Cell. Biol. 19, (1999). 3. Harrison, D. E. Immunogenetics 13, (1981). 4. Kitamura, Y., Go, S. & Hatanaka, K. Blood 52, (1978). 5. Waskow, C., Bartels, S., Schlenner, S. M., Costa, C. & Rodewald, H. R. Blood 109, (2007). 6. Rodewald, H. R., Ogawa, M., Haller, C., Waskow, C. & DiSanto, J. P. Immunity 6, (1997). 7. Madan, V. et al. Blood 113, (2009).