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. 2018 Feb 27;115(9):2180-2185.
doi: 10.1073/pnas.1718446115. Epub 2018 Jan 31.

Respecifying human iPSC-derived blood cells into highly engraftable hematopoietic stem and progenitor cells with a single factor

Yu-Ting Tan  1   2 , Lin Ye  2 , Fei Xie  2 , Ashley I Beyer  3 , Marcus O Muench  3   4 , Jiaming Wang  2 , Zhu Chen  1 , Han Liu  5 , Sai-Juan Chen  5 , Yuet Wai Kan  6   4   7
Affiliations

Respecifying human iPSC-derived blood cells into highly engraftable hematopoietic stem and progenitor cells with a single factor

"V体育安卓版" Yu-Ting Tan et al. Proc Natl Acad Sci U S A. .

Abstract

Derivation of human hematopoietic stem cells (HSCs) from induced pluripotent stem cells (iPSCs) offers considerable promise for cell therapy, disease modeling, and drug screening. However, efficient derivation of functional iPSC-derived HSCs with in vivo engraftability and multilineage potential remains challenging VSports手机版. Here, we demonstrate a tractable approach for respecifying iPSC-derived blood cells into highly engraftable hematopoietic stem and progenitor cells (HSPCs) through transient expression of a single transcription factor, MLL-AF4 These induced HSPCs (iHSPCs) derived from iPSCs are able to fully reconstitute the human hematopoietic system in the recipient mice without myeloid bias. iHSPCs are long-term engraftable, but they are also prone to leukemic transformation during the long-term engraftment period. On the contrary, primary HSPCs with the same induction sustain the long-term engraftment without leukemic transformation. These findings demonstrate the feasibility of activating the HSC network in human iPSC-derived blood cells through expression of a single factor and suggest iHSPCs are more genomically instable than primary HSPCs, which merits further attention. .

Keywords: MLL-AF4; engraftability; hematopoietic stem cells; human induced pluripotent stem cells; leukemia. V体育安卓版.

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Conflict of interest statement (VSports在线直播)

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
In vitro induction of MLL-AF4 in iPSC-derived hematopoietic cells. (A) The scheme of in vitro differentiation of iPSCs into hematopoietic cells. (B) Morphological change of iPSCs during the in vitro differentiation. (Scale bar, 1 μm.) (C) Colony-forming assay of iPSC-derived hematopoietic cells harvested at day 12 of in vitro differentiation. BFU-E, burst-forming unit-erythroid; GEMM, colony-forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte; GM, colony-forming unit-granulocyte, macrophage; G, colony-forming unit-granulocyte. All are shown at 40× magnification. (D) Phenotypic analysis of iPSC-derived hematopoietic cells harvested at day 12 of in vitro differentiation. (E) The serial replating potential of iPSC-derived hematopoietic cells with or without (w/o) Dox induction. FACS-sorted GFP+ and GFP− cells that were immediately seeded on methocult were labeled as “w/o DOX” group. Data are shown as mean ± SD of three independent experiments. (F) Flow cytometry analysis of MLL-AF4–induced iPSC-derived hematopoietic cell harvested at day 12 of in vitro differentiation. MLL-AF4 and rtTA plasmids were transfected followed by 72-h induction of MLL-AF4 with the addition of 2 μg⋅ml−1 doxycycline. (G) Principle component analysis of RNA-Seq data from in vitro-derived HSPCs from iPSC (iPSC-HSPCs, CD34 iPSC, or MN iPSC derived) without MLL-AF4 transfection (CD34_w/o MA4; MN_w/o MA4) or with MLL-AF4 transfection (CD34 + MA4; MN + MA4), compared with the peripheral blood mobilized CD34+ HSC (mobHSC), and the publicly available dataset for common myeloid progenitor (CMP) and lymphoid-primed multipotent progenitor (LMPP). (H) GSEA signatures of in vitro-derived iPSC-HSPCs without MLL-AF4 transfection (w/o MA4) or with MLL-AF4 transfection (+MA4). P < 0.05 and false discovery rate (FDR) <0.25 were considered significant conditions. (I) Heat map showing relative gene expression of regulatory genes identified as HSC specific in the indicated cell types.
Fig. 2.
Fig. 2.
In vivo induction of MLL-AF4 in iPSC-derived hematopoietic cells enables potent engraftment and multilineage reconstitution. (A) The scheme of transplanting induced iPSC-HSPCs into newborn NSG mice. MA4, MLL-AF4; in vitro diff, in vitro differentiation; BM, bone marrow; PB, peripheral blood. (B) The analysis of human cell chimerism in the bone marrow of recipient mice after 8 wk of transplantation. Transplant groups are as described in A. NT, nontransfection control. (C) Multilineage reconstitution of MLL-AF4 plasmid-treated iPSC-HSPCs in the BM of recipient mice at 8 wk posttransplant. (D) Multilineage reconstitution of human erythroid cells (E; CD235a+ or CD71+), myeloid cells (M; CD33+), B cells (B; CD19+), and T cells (T; CD3+) in the BM, spleen and PB of engrafted mice transplanted with MLL-AF4 plasmid transfected iPSC-HSPCs at 8 wk posttransplant. (E) Total chimerism of human CD45+ cells in the harvested tissues of engrafted mice for each transplant group at 8 wk posttransplant. LV, lentivirus. Data were analyzed for two independent experiments with three mice each time and shown as mean ± SD *P < 0.05.
Fig. 3.
Fig. 3.
iHSPCs but not primary HSPCs undergo the leukemic transformation during the long-term engraftment period. (A) Chimerism of human cells in the bone marrow of recipient mice over 16 wk posttransplant. (B) Phenotypic analysis of engrafted human cells in the bone marrow (BM) of recipient mice with long-term engraftment. Gated human CD45+ cells were analyzed for myeloid (CD33) and lymphoid (CD10) (Mye/Lym) lineage distribution, percentage of progenitor (CD34+CD38−, CD7+CD45RA+), and B cell (CD19) and T cell (CD3) compartments (B/T). (C) Engraftment of primary HSPCs with (+MA4) or without (w/o MA4) transfection of MLL-AF4 in the peripheral blood (PB), spleen, and BM of recipient mice after 12–16 wk of transplantation. (D) Phenotypic comparison of engrafted human CD45+ cells in the BM of MLL-AF4 transfected transplants or nontransfected transplants. (E) Profiling of B cell leukemia-associated mutations in the in vitro-derived iPSC-HSPCs without MLL-AF4 transfection (CD34 vitro_w/o MA4; MN vitro_w/o MA4) or with MLL-AF4 transfection (CD34 vitro_MA4; MN vitro_MA4), and in vivo-derived human CD45+ cells engrafted in the BM of primary recipients over 9–16 wk posttransplant with MLL-AF4 transfection. CD34_wk9, CD34-iPSC derived HSPCs transfected with MLL-AF4 were transplanted and harvested for human CD45+ cells at 9 wk posttransplant; the same meaning for MN-iPSC group. HSPC + MA4_wk16, BM cells harvested from engrafted recipients transplanted with primary HSPCs with MLL-AF4 induction at 16 wk posttransplant; MNCs, peripheral blood-derived mononuclear cells; mobHSC, mobilized HSCs. (F) PCA analysis of RNA-Seq data from in vivo-derived human CD45+CD34+ cells in the bone marrow of primary recipient mice transplanted with MLL-AF4 transfected primary HSPCs at 16 wk posttransplant (MA4 treated HSPC_vivo), compared with the MLL-AF4 transfected iPSC-HSPCs (CD34-iPSC derived) before transplantation (MA4-treated iPSC-HSPC_vitro) or 16 wk posttransplant (MA4-treated iPSC-HSPC_vivo), and mobilized HSC (mobHSC). (G) GO analysis of in vivo-derived human CD45+ cells from engrafted iHSPCs (16 wk posttransplant) compared with the in vitro-derived iPSC-HSPCs. Significantly deregulated genes enriched in hematopoietic cell lineage signature were indicated. (H) GO analysis of enriched features of MLL-AF4 transfected primary HSPCs compared with MLL-AF4 transfected iPSC-HSPCs. In vivo-derived cells of both groups were harvested at 16 wk posttransplant. Gray bars indicate the enriched features containing genes that are mostly down-regulated, and the opposite for the black bars. All RNA-Seq data were generated from three independent biological replicates and analyzed by the average FPKM (fragments per kilobase of transcript per million mapped reads) value.
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References (VSports在线直播)

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