COMMENTARY COMMENTARY

MLL-AF4, a double-edged sword for iPSC respecification into HSPCs Bernd B. Zeisiga and Chi Wai Eric Soa,1

The successful derivation of human embryonic stem (MLL-AF4) to respecify iPSCs in- cells (hESCs) in 1998 (1) and later of induced pluripo- to HSPCs in vivo, capable of multilineage engraftment tent stem cells (iPSCs) in 2006/2007 (2, 3) led to huge (Fig. 1). The authors generated iPSCs from human pe- excitement and held great promise to revolutionize ripheral blood mobilized HSPCs (CD34-iPSCs) and several fields, from basic research to regenerative mononuclear cells (MN-iPSCs), and then applied a and personalized medicine, as these human pluripo- feeder-free and xeno-free culture condition for differ- + tent cells (hPSCs) have the potential when cultured entiation of iPSCs toward hematopoietic cells (CD34 + under the right conditions to give rise to every line- CD45 ) that were, however, predominantly of myeloid + age, tissue, and cell of the human body. Given the phenotype (CD33 ). Realizing that many of the pre- unique source and properties of these cells, the re- viously described transcription factors with the poten- search as well as clinical uses of hESCs and iPSCs tial to respecify HSPCs (10, 11) are closely correlated were, however, from the beginning met with a com- with the mixed lineage leukemia gene (MLL) or MLL mon and a unique set of ethical and safeguarding fusions, the authors opted to use doxycycline (DOX)- issues, which are still being hotly debated (4–6). More- inducible MLL-AF4 [the product of the t(4:11) chromo- over, protocols for efficient differentiation of hPSCs somal translocation frequently found in infant B cell into the desired tissues and cell types still need sub- acute lymphoblastic leukemia (B-ALL)] to assess its stantial optimizations for efficient and safe clinical ability to convert iPSC-derived blood cells into HSPCs. uses. As a result, despite the enthusiasm and intensive Interestingly, iPSC-derived blood cells with activated research activities, the clinical application of hPSCs is MLL-AF4 not only showed a higher degree of replat- still in its early phases and needs to overcome various ing ability compared with those without DOX treat- obstacles for its full potential (4–6). One of the major ment, but also displayed a marked increase in bottlenecks for its hematopoietic application is the (ex lymphoid marker CD10 expression (more than 10% + vivo) generation of functional human hematopoietic of CD45 cells), suggesting that MLL-AF4 could convey stem/progenitor cells (HSPCs) with long-term repopu- self-renewal and lymphoid differentiation potential. lating capacity and multilineage differentiation poten- RNA sequencing (RNA-seq) on MLL-AF4 transduced tial (7, 8). Not until recently has a protocol for in vivo iPSC-derived blood cells at day 4 of DOX induction respecification of iHSPCs been reported (9), where was performed. Principal-component analysis placed hESCs were subjected to morphogen-directed differenti- the MLL-AF4 transduced cells apart from the nontrans- ation into hemogenic (HE), which then were duced cells and in close proximity to mobilized primary cultured under endothelial-to-hematopoietic transition HSPCs. Gene set enrichment analysis (GSEA) further conditions followed by transient expression of seven tran- revealed expression programs associated with self- scriptionfactors(ERG,HOXA5,HOXA9,HOXA10,LCOR, renewal and definitive hematopoiesis being conferred RUNX1, and SPI1). Upon transplantation, long-term and by MLL-AF4. When transplanted into newborn NSG im- transplantable multilineage (B, T, and myeloid cells) en- munocompromised mice, untransduced iPSC-derived graftment was observed, albeit with notable B cell bias blood cells only showed 0.18% engraftment after 8 wk. (Fig. 1) (9). Despite the requirement of an intermediate Conversely, engraftment of MLL-AF4 transduced cells, cell state and expression of multiple factors, this study where DOX was administered for 3 d in vitro and 2 wk represents a major step toward the clinical application in vivo, could be detected up to around 20% in the bone of hESCs for generation of human HSPCs. marrow. Importantly, this engraftment was multilineage, In PNAS, Tan et al. (10) describe a simplified and with myeloid, B, and T cells present in bone marrow, efficient differentiation protocol without the requirement spleen, and peripheral blood of analyzed mice. Interest- of an intermediate cell state (e.g., HE) by using a single ingly, transduction with five transcription factors (11)

aLeukaemia and Stem Cell Biology Group, School of Cancer and Pharmaceutical Sciences, King’s College London, London SE5 9NU, United Kingdom Author contributions: B.B.Z. and C.W.E.S. wrote the paper. The authors declare no conflict of interest. Published under the PNAS license. See companion article on page 2180. 1To whom correspondence should be addressed. Email: [email protected]. Published online February 14, 2018.

1964–1966 | PNAS | February 27, 2018 | vol. 115 | no. 9 www.pnas.org/cgi/doi/10.1073/pnas.1800622115 Downloaded by guest on September 24, 2021 A Schemac protocol used by Sugimura et al. hESC-derived hESC HE blood cells 2 weeks DOX in vivo + 7 TFs

Culture Culture Respecificaon 8 days 3 days into iHSPCs in vivo adult NSG CD34+CD43- CD34+CD45+ are emerging Schemac protocol used by Tan et al. iPSC-derived iPSCs blood cells 2 weeks DOX in vivo +MLL-AF4

Culture Respecificaon 12 days into iHSPCs in vivo Newborn NSG CD34+CD45+

B Summary of in vivo iHSPC properes Sugimura et al. Tan et al.

Mul-lineage engrament in primary recipients ? Yes Yes Transplantable? Yes Yes Lineage-balanced engrament in secondary recipients? No No Prone to B-cell transformaon? Not reported Yes

Fig. 1. Schematic representation of the different strategies used to date to generate functional human induced hematopoietic stem/progenitor cells (iHSPCs) with long-term multilineage engraftment potential. (A) Scheme of the protocol used by Sugimura et al. (9) (Top) and that used by Tan et al. (10) (Bottom) for the generation of human pluripotent stem cell (hPSC)-derived iHSPCs. DOX, Doxycycline; HE, hemogenic endothelium; 7 TFs, seven transcription factors (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, and SPI1). (B) Tabular summary of the key in vivo features of iHSPCs.

alone resulted in only modest engraftment levels of around 0.40%, To rule out that the current protocol generally favors leukemic and their expression in addition to MLL-AF4 did not further increase transformation, identical transplantation experiments were also + the MLL-AF4 engraftment levels. Long-term observation (16 wk) of carried out using primary HSPCs (mobilized CD34 HSPCs iso- engrafted mice showed continuous increase of donor cells in the lated from peripheral blood). As expected, while untransduced bone marrow, but there was a significant shift toward lymphoid cell primary HSPC could engraft recipient mice, the engraftment populations at the expense of myeloid cells in the bone marrow, levels were higher in the MLL-AF4 group. However, MLL- spleen, and peripheral blood. Further characterization of the trans- AF4 did not alter the lineage distribution in primary as well as planted animals revealed splenomegaly and a high percentage in secondary transplanted mice compared with untransfected of cells with cellular morphology of lymphoblasts with abnormal primary HSPC transplanted mice. In line with previous find- high nuclear/cytoplasmic ratio, suggesting that B cell leukemia ings (12), MLL-AF4 primary HSPC transplanted mice did not transformation had taken place. This leukemic phenotype was also show splenomegaly or developed leukemia. Bone marrow of transplantable into secondary recipients. Importantly, no MLL- engrafted mice exhibited diverse and normal cellular morphol- AF4 expression was detected in any of the leukemic samples, which ogies, suggesting that the current experimental scheme of instead carried point mutations, fusion transcripts (two of six mice had expressing MLL-AF4 did not universally drive the transformation BCR-ABL), and other genomic aberrations usually associated with of targeted cells. Interestingly, RNA-seq revealed that random ALL. These results suggest that, instead of MLL-AF4, other driver point mutations are present in genes involved in B-ALL only in mutations as a result of genomic instability may sustain the disease. the leukemic samples but were absent from in vitro cultured

Zeisig and So PNAS | February 27, 2018 | vol. 115 | no. 9 | 1965 Downloaded by guest on September 24, 2021 iHSPCs (with or without MLL-AF4) and MLL-AF4 transduced pri- cells acquire mutations during reprogramming, culture, and dif- mary HSPCs that were harvested after a 16-wk in vivo period. ferentiation that may affect their laboratory and clinical utility (13– GSEA consistently indicated a more stable genetic status in MLL- 16). Moreover, given the transient nature of MLL-AF4 expression, AF4 primary HSPCs compared with MLL-AF4 iHSPCs. Finally, and the experimental evidence that MLL-leukemia is addicted to analysis further revealed that most enriched mo- the MLL fusion (17–19), it seems plausible that genomic instability lecular features in in vivo-derived leukemic cells were related to of iPSCs rather than transient expression of MLL-AF4 is a major cancer and B-ALL, compared with in vitro iHSPCs or engrafted factor in leukemic transformation of iPSCs in this study, although primary HSPCs. MLL-AF4 may have helped to initiate the process. Regardless, the As production of sufficient numbers of functional hematopoi- development of B cell leukemia has severely limited the application etic (stem) cells has been one of the major challenges for the use of this protocol in its current form, unless further modifications can of hPSCs in experimental as well as clinical settings, the develop- eliminate or minimize this important caveat. It is notable that viral ment of simple and efficient methods for generation of functional expression of MLL-AF4 in hESCs using a different protocol does not differentiated cells from human iPSC/ES cells is a holy grail in the lead to their leukemic transformation (20), which may suggest that field. Therefore, this study, which is based on transient expression hESCs are more genetically and epigenetically stable than iPSCs. It of a single transcription factor in human iPSCs without going is tempting to test the same or similar protocol in hESCs to see through a cellular intermediate, resulting in a very significant whether a similar level of respecification can be achieved without ∼ 20% in vivo contribution of multiple hematopoietic lineages, is leukemia induction. On the other hand, further adjustments of the an important work and sheds light into the possible development current protocol, such as shortening the transient expression time of a much simplified iPSC differentiation protocol for the genera- of the MLL fusion or replacing it with wild-type or other forms of tion of iHSPCs (Fig. 1). However, the observed in vivo leukemic MLL (fusions), could be of interest for future studies. Nevertheless, transformation phenotype in the present study also highlights the current study represents an important step for the development potential caveats for the use of iPSC technology. The acquisition of a much simplified protocol for generation of iHSPCs and at the of mutations over time in vivo in MLL-AF4 iHSPCs but not MLL- same time highlights the challenges that we are facing. AF4 primary HSPCs strongly suggests that epigenetic and tran- scriptional plasticity and the preferential use of error-prone DNA Acknowledgments damage pathways might represent potential issues for the clinical This work was supported by programme grants from Cancer Research UK use of iPSCs. Indeed, there is a large body of evidence that iPSC and Bloodwise.

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