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S. Singbrant et al.
While fluorescence-activated cell sorting (FACS)-based methods for fractionation of distinct erythroid progenitor cells in murine and human during steady-state8-11 has enabled in-depth characterization of mechanisms regulating steady-state erythropoiesis,11-15 the cells and mechanisms regulating stress-erythropoiesis remain poor- ly defined. To enable studies of stress-erythropoiesis we set out to identify novel marker-combinations separating and enriching for the early stress-progenitors mediating radioprotection and recovery from severe anemia. We pre- viously demonstrated that fetal erythroid burst-forming- units (BFU-E) can be isolated as lineage- cKit+CD71/CD24alowSca1–CD34– with high purity from murine fetal liver, where erythropoiesis in many ways resemble stress-erythropoiesis.16 Attempts by other groups to isolate adult stress-erythroid progenitors from spleens of anemic mice and in vitro cultures have shown stress-BFU-E to be lineage-cKit+CD71/Ter119low, and fur- ther enriched in the Sca1+CD34–CD133– fraction. However, very few of these cells possess BFU-E potential (0.1-0.2%). Furthermore, in the active debate on lineage potential of stem- and progenitor cells, genuine megakary- ocytic/erythroid potential is often overlooked since mature erythrocytes and platelets are difficult to trace in vivo after transplantation. Hence, the identity of pure stress-BFU-E remains largely elusive.
Using a novel combination of surface markers together with the tracing marker Kusabira Orange which is expressed in all cells, we have developed a method for high purity fractionation of a hierarchy of multi-potent progenitors, stress-BFU-E, and stress-CFU-E within the lineage-cKit+CD71/CD24alow cells in spleen during irradia- tion-induced stress-erythropoiesis as well as in steady- state BM, and for the first time determined their kinetics and full differentiation potential in vivo. The formation of stress-BFU-E was highly dependent on functional BMP- signaling, and stress-BFU-E displayed enhanced expres- sion of BMP-responsive genes, as well as gene signatures associated with erythropoiesis and proliferation compared to their steady-state counterpart. In addition, discrepan- cies in the epigenetic landscape were selectively enriched for putative binding sites for the chromatin-looping tran- scription factor CTCF. In conclusion, our findings provide high-purity isolation of both steady-state BFU-E and the stress-BFU-E mediating recovery from severe anemia, and offer molecular insight to and functional determination of the unique capacity of stress-BFU-E to rapidly form ery- throid cells in response to anemia.
Methods
Mice and transplantations
All procedures involving mice were approved by the Animal Ethics Committee of Malmö/Lund, Sweden. Anemia was induced by lethally irradiating 8-12 week-old recipient mice (C57Bl/6; Ly5.2) with a split dose of 2x500 cGy, followed by transplantation of 2x106 unfractionated BM cells to rescue and trigger stress-ery- thropoiesis. Donor mice (B6SJL; Ly5.1) were either Kusabira Orange (KuO) positive or negative. Recipients were sacrificed on day 8 for analysis of stress recovery in the spleen.
For in vivo tracing, 500 multipotent progenitors (sMPP), 5,000 sBFU-E or 5,000 sCFU-E, all KuO+, were FACS-sorted from day 8 stressed spleens and transplanted into lethally irradiated secondary recipients together with 105 unfractionated wild-type
BM cells as support. Secondary recipients were bled at 1, 2 and 4 weeks, and sacrificed at 2 or 4 weeks post transplantation for analysis of lineage potential and kinetics in peripheral blood (PB), BM and spleen.
Flow cytometry
A complete description of all antibodies used is listed in the Online Supplementary Materials and Methods.
Hematopoietic progenitor assays
All colony assays were incubated at 37°C incubators in 5% CO2 with either 21% or 1-4% O2 as indicated, and scored on day 4 (CFU-E) or 7-8 (BFU-E and mixed colonies).
RNA sequencing
Splenic stress- and BM steady-state progenitors were FACS- sorted in triplicates. Strand specific RNA-sequencing libraries were constructed using SMARTer Stranded Total RNA-Seq Kit v2 (Takara Bio) followed by sequencing on a HiSeq3000 (Illumina).
Assay for Transposase Accessible Chromatin sequencing
3,000 splenic stress- and BM steady-state BFU-E were FACS- sorted in triplicates for the assay of Transposase Accessible Chromatin (ATAC) library preparation and sequencing as described previously.18 Libraries were subject to single-end sequencing on a NextSeq500 (Illumina).
Statistical analysis
For all statistical analysis, apart from RNA- and ATAC-sequencing for which the specifications are stated in the Online Supplemental Materials and Methods, statistical significance was calculated using ANOVA accounting for multiple compar- isons, followed by Tukey's multiple comparisons test. One-way ANOVA was used for single time point analysis and Two-way ANOVA was used when measuring potential over time (Figure 2). *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.
A detailed description of all methods used is available online in the Online Supplementary Materials and Methods.
Results
CD150, CD9 and Sca1 identify a hierarchy of splenic stress-progenitors during irradiation-induced stress recovery
To identify the stress-progenitors involved in irradia- tion-induced anemia and stress recovery, we subjected mice to lethal irradiation followed by BM transplantation and analyzed recipient spleens on day 8 (Figure 1A) when the greatest expansion of stress-progenitors occurs.3 Using this model, stress-progenitors were previously identified as Lin–cKit+CD71/Ter119low, although at low frequencies (0.2%).3 To further enrich for stress-erythroid progenitors we included CD150, known to mark megakaryocytic/ery- throid progenitors during steady-state hematopoiesis.9 All BFU-E potential in the cKit+CD71low/Ter119low population resided in the CD150+ fraction (Figure 1B-C). Extramedullary expansion of early erythroid progenitors during stress-erythropoiesis is reminiscent of fetal liver erythropoiesis. We therefore analyzed previously pub- lished mRNA expression data (GSE26086)16 from E14.5 to E15.5 fetal liver erythroid progenitors to identify possible additional markers for further sub-division of adult stress- erythroid progenitors (Online Supplementary Figure S1A).
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haematologica | 2020; 105(11)