Disruption of normal stem cell function and transmission of myelodysplastic syndrome by self-renewal of committed myeloid lineage cells

Abstract

The ineffective hematopoiesis of myelodysplastic syndrome (MDS) suggests that hematopoietic stem and progenitor cells (HSPCs) are defective. Here, we demonstrate that NUP98::HOXD13 (NHD13) MDS mice have significantly decreased functional HSPCs. Moreover, in contrast to wild-type (WT) bone marrow (BM), lineage-positive (Lin⁺) BM cells from NHD13 mice have self-renewal potential. Specific subsets of NHD13 Lin⁺ cells that express B220 and Kit antigens were able to self-renew and generate MDS in WT recipients. Although this unique B220⁺Kit⁺ phenotype could be found in WT as well as NHD13 BM, the population was markedly increased in NHD13 BM. Further characterization using Mac1 and Gr1 markers revealed that both Mac1⁺Gr1⁺B220⁺Kit⁺ and Mac1^-Gr1^- B220⁺Kit⁺ populations showed self-renewal and led to an MDS phenotype in WT recipients. Taken together, these findings demonstrate that as normal hematopoiesis derived from typical HSPCs decreases in NHD13 mice, committed hematopoietic progenitor cells proliferate, self-renew, and initiate MDS.

Keywords: MDS-initiating cell; NHD13; hematopoiesis; kit; myelodysplastic syndrome; stem cells.

Graphical abstract

Figure 1

Hematopoietic abnormalities in NHD13 mice progress with age (A) Complete blood count (CBC) from age-matched WT (n = 9) and NHD13 (n = 9) mice: ^∗< p = 0.05; ^∗∗< p = 0.01. (B) HSPC analysis from age-matched WT (n = 9) and NHD13 (n = 9) mice: ^∗< p = 0.05; ^∗∗< p = 0.01. (C) Numbers of CD41 N (negative) LT-HSCs at 2 and 6 months of age: WT n = 4, NHD13 n = 4; LN, lineage negative; MPP, multi-potential progenitor; 2F2T, two femora and two tibiae per mouse; ^∗< p = 0.05; ^∗∗∗< p = 0.001.

Figure 2

Engraftment of purified HSPC subpopulations from NHD13 bone marrow (A) Diagram of the experiment and cell sorting. (B) Engraftment of CD45.2⁺NHD13 cells at indicated time post transplant: MPP4 n = 5, MPP3 n = 5, MPP2 n = 5, ST-HSCs n = 4, LT-HSCs n = 3. (C) Donor cell engraftment assessed by PCR. Genomic DNA prepared from BM cell aspirated from femur of recipient mice at post-transplantation week 17; PC, positive control for NHD13 transgene; NTC, no template control; ^∗ indicates non-specific band, likely primer dimers. The results shown represent one of two independent experiments. (D) Two independent experiments, HSCT of 200 LSK cells from either WT or NHD13 BM: WT LSK_200 n = 4, NHD LSK_200 n = 5.

Figure 3

Engraftment and multi-lineage potential of NHD13 Lin⁺ BM cells (A) Schematic outline of the experiment. Four- to 6-month-old NHD13 or WT mice served as donors. (B–E) Peripheral blood engraftment of Lin⁻ (LN) or Lin⁺ (LP) cells from WT or NHD13 donors. Results represent individual mice from four independent experiments (designated by experiment number and mouse number); blue lines indicate engraftment from WT LN or LP donor cells. Dark red lines indicate engraftment from NHD13 LN or LP donor cells. (F) Percentage of myeloid and lymphoid cells at 24 and 40 weeks post transplant (PT): M+, Mac1 single positive; M+ G+, Mac1 and Gr1 double-positive; Lym, lymphoid. (G) Percent blast cells in NHD13 Lin⁺ BM cells at time of euthanasia. (H) Morphology of CD45.2-purified BM cells from NHD13 LP BM recipients. BM cells were purified using magnetic cell sorting (MACS) with a CD45.2 antibody and stained with May-Giemsa (MG) stain (400X); arrowhead, blast cell; arrow, ring neutrophil. (I) Percent chimerism of NHD13 cells in NHD13 LP BM recipients. (J) Summary of differentiated progeny of the NHD13 Lin⁺ BMCs in recipient mice (n = 3). (K) Detection of the NHD13 transgene in flow-sorted populations. Amplification of the Scid locus is used as a DNA quality control; decreased intensity of the Scid PCR product is expected for erythroid, T, and B cells as fewer cells were recovered after sorting.

Figure 4

Distinct repopulation patterns from NHD13 Lin⁺ or Lin⁻ BM cells (A) Schematic of the experiment. (B) Representative flow cytometry profiles from NHD13 LN recipient. (C) Representative flow cytometry profiles from NHD13 LP recipient. (D) Percent LN BM cells in LN (n = 4) vs. LP (n = 3) recipients: ^∗∗< p = 0.01. (E) HSPC repopulation in each transplant group: NHD13 n = 3, WT n = 3 in NHD13 LP recipients; NHD13 n = 4, WT n = 4 in NHD13 LN recipients; ^∗∗∗< p = 0.001.

Figure 5

Distinct long-term repopulation features of NHD13 Lin⁺ vs. Lin⁻ BM cells (A) Secondary recipients of NHD13 LN BM cells: LN4#1 n = 5, LN4#4 n = 5, LN5#2 n = 4. (B) Secondary recipients of NHD13 LP BM cells: LP3#1 n = 3, LP3#2 n = 5, LP4#3 n = 4. (C) Short-term (6 weeks) engraftment of primary (upper: LP BM n = 5, LN BM n = 6) or secondary (lower: LP BM n = 9, LN BM n = 14) recipients of NHD13 LN or LP BM cells: ^∗< p = 0.05; ^∗∗∗< p = 0.001. (D) AML incidence in secondary recipients following transplantation. Results from three independent experiments. LP or LN-2° indicates secondary recipients of NHD13 LP or LN BM, respectively. (E and F) Summary of whole-exome sequencing (WES) results. The digits in the brackets indicate variant allele frequencies.

Figure 6

MDS-initiating cell populations in NHD13 Lin⁺ BM (A) Outline of the experiment. (B) Engraftment kinetics and estimated frequency of MIC in each sorted cell population. LPK+, positive for lineage makers and Kit (n = 4); M+ G+, positive for both Mac1 and Gr1 (n = 3). (C) Comparison of Mac1, Gr1, B220, and Kit staining of WT and NHD13 BM cells. (D) Proportion of M+G+B+K+ (Mac1⁺Gr1⁺B220⁺Kit⁺) and M−G−B+K+ (Mac1⁻Gr1⁻B220⁺Kit⁺) cells in NHD13 (n = 9) and WT BM (n = 7): ^∗< p = 0.05; ^∗∗∗< p = 0.001. (E) Each bar represents the proportion of recipient mice engrafted with CD45.2 cells (≥0.5%) in PB at the indicated time. Results analysis from seven independent experiments. (F) Mean percent engraftment of engrafted recipients at the indicated time. Each number of recipients is the same as indicated in the graph (E) legend. Results from seven independent experiments. (G) Representative flow cytometry profiles for recipients of NHD13 M−G−B+K and M+G+B+K+ cells. (H) Summary of repopulation from NHD13: M+G+B+K+ n = 7, M−G−B+K+ n = 7 in the left; M+G+B+K+ n = 6, M−G−B+K+ n = 6 in the right; ^∗∗< p = 0.01.