Patient-Derived iPSCs Faithfully Represent the Genetic Diversity and Cellular Architecture of Human Acute Myeloid Leukemia.

The reprogramming of human acute myeloid leukemia (AML) cells into induced pluripotent stem cell (iPSC) lines could provide new faithful genetic models of AML, but is currently hindered by low success rates and uncertainty about whether iPSC-derived cells resemble their primary counterparts. Here we developed a reprogramming method tailored to cancer cells, with which we generated iPSCs from 15 patients representing all major genetic groups of AML. These AML-iPSCs retain genetic fidelity and produce transplantable hematopoietic cells with hallmark phenotypic leukemic features. Critically, single-cell transcriptomics reveal that, upon xenotransplantation, iPSC-derived leukemias faithfully mimic the primary patient-matched xenografts. Transplantation of iPSC-derived leukemias capturing a clone and subclone from the same patient allowed us to isolate the contribution of a FLT3-ITD mutation to the AML phenotype. The results and resources reported here can transform basic and preclinical cancer research of AML and other human cancers. SIGNIFICANCE: We report the generation of patient-derived iPSC models of all major genetic groups of human AML. These exhibit phenotypic hallmarks of AML in vitro and in vivo, inform the clonal hierarchy and clonal dynamics of human AML, and exhibit striking similarity to patient-matched primary leukemias upon xenotransplantation. See related commentary by Doulatov, p. 252. This article is highlighted in the In This Issue feature, p. 247.

Feasibility of early activation after cochlear implantation.

OBJECTIVES: The purpose of the study is to investigate feasibility of early activation after cochlear implantation by evaluating long-term impedance change and speech perception. DESIGN: Case-control study SETTING: Between July 2015 and December 2016, we prospectively enrolled 20 subjects for early activation (within 24 hours after cochlear implantation). On the other hand, from November 2013 to July 2015, 20 age- and sex-matched control subjects from the database of cochlear implantees treated with conventional activation schedule (4 weeks after surgery) were retrospectively enrolled. PARTICIPANT: Forty patients who underwent cochlear implantation surgeries. MAIN OUTCOME MEASURES: The series impedance and speech perception score of both groups were compared. RESULTS: No statistical difference in long-term follow-up between the two groups was found using GEEs and multivariate analysis. In the early activation group, impedance reached a steady level by the 2nd postoperative week, and the hearing perception ability significantly improved by the 4th postoperative week. CONCLUSION: This comparative study illustrated sequential impedance data during early activation (24 hours) and conventional activation (4 weeks) after CI surgery. There were no major complications in either group, and the safety of early activation with respect to impedance changes, postoperative residual hearing preservation and speech perception scores were non-inferior to that of the conventional group. Therefore, in this study, we established the feasibility of early activation 24 hours after cochlear implantation.

Treatment of moderate-to-severe otosclerosis with simultaneous piston surgery and incus vibroplasty.

OBJECTIVE: Whereas the nature of otosclerosis has been extensively investigated, treatment modalities in advanced otosclerosis with the sensorineural hearing loss (SNHL) are relatively unexplored. MATERIALS AND METHODS: This article presents a retrospective case series study of nine patients who received a one-stage piston coupled with Vibrant Soundbridge® vibroplasty in treating otosclerosis with moderate-to-severe SNHL. RESULTS: The findings suggest that hearing loss could be restored across frequencies and no significant change in the bone-conduction threshold were measured. CONCLUSION: One-stage piston surgery coupled with incus vibroplasty is a safe procedure and has sufficient efficacy to restore hearing loss in patients with otosclerosis with moderate-to-severe SNHL.

Navigation-assisted endoscopic endonasal surgery of a glomangiopericytoma with intraorbital extension: A case report and literature review.

A glomangiopericytoma, or sinonasal type hemangiopericytoma, is a rare lesion which accounts for <0.5% of all sinonasal tumors. The mainstay treatment is wide excision. Instead of traditional open surgical approaches, such as midfacial degloving or lateral rhinotomy, we offer a case of 21-year-old male with diagnosis of glomangiopericytoma with skull base and intraorbital invasion and received navigation-assisted endoscopic excision of a glomangiopericytoma.

Dissecting the Contributions of Cooperating Gene Mutations to Cancer Phenotypes and Drug Responses with Patient-Derived iPSCs.

Connecting specific cancer genotypes with phenotypes and drug responses constitutes the central premise of precision oncology but is hindered by the genetic complexity and heterogeneity of primary cancer cells. Here, we use patient-derived induced pluripotent stem cells (iPSCs) and CRISPR/Cas9 genome editing to dissect the individual contributions of two recurrent genetic lesions, the splicing factor SRSF2 P95L mutation and the chromosome 7q deletion, to the development of myeloid malignancy. Using a comprehensive panel of isogenic iPSCs-with none, one, or both genetic lesions-we characterize their relative phenotypic contributions and identify drug sensitivities specific to each one through a candidate drug approach and an unbiased large-scale small-molecule screen. To facilitate drug testing and discovery, we also derive SRSF2-mutant and isogenic normal expandable hematopoietic progenitor cells. We thus describe here an approach to dissect the individual effects of two cooperating mutations to clinically relevant features of malignant diseases.

TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells.

TET enzymes oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which can lead to DNA demethylation. However, direct connections between TET-mediated DNA demethylation and transcriptional output are difficult to establish owing to challenges in distinguishing global versus locus-specific effects. Here we show that TET1, TET2 and TET3 triple-knockout (TKO) human embryonic stem cells (hESCs) exhibit prominent bivalent promoter hypermethylation without an overall corresponding decrease in gene expression in the undifferentiated state. Focusing on the bivalent PAX6 locus, we find that increased DNMT3B binding is associated with promoter hypermethylation, which precipitates a neural differentiation defect and failure of PAX6 induction during differentiation. dCas9-mediated locus-specific demethylation and global inactivation of DNMT3B in TKO hESCs partially reverses the hypermethylation at the PAX6 promoter and improves differentiation to neuroectoderm. Taking these findings together with further genome-wide methylation and TET1 and DNMT3B ChIP-seq analyses, we conclude that TET proteins safeguard bivalent promoters from de novo methylation to ensure robust lineage-specific transcription upon differentiation.

Stage-Specific Human Induced Pluripotent Stem Cells Map the Progression of Myeloid Transformation to Transplantable Leukemia.

Myeloid malignancy is increasingly viewed as a disease spectrum, comprising hematopoietic disorders that extend across a phenotypic continuum ranging from clonal hematopoiesis to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In this study, we derived a collection of induced pluripotent stem cell (iPSC) lines capturing a range of disease stages encompassing preleukemia, low-risk MDS, high-risk MDS, and secondary AML. Upon their differentiation, we found hematopoietic phenotypes of graded severity and/or stage specificity that together delineate a phenotypic roadmap of disease progression culminating in serially transplantable leukemia. We also show that disease stage transitions, both reversal and progression, can be modeled in this system using genetic correction or introduction of mutations via CRISPR/Cas9 and that this iPSC-based approach can be used to uncover disease-stage-specific responses to drugs. Our study therefore provides insight into the cellular events demarcating the initiation and progression of myeloid transformation and a new platform for testing genetic and pharmacological interventions.

Amelioration of Hyperbilirubinemia in Gunn Rats after Transplantation of Human Induced Pluripotent Stem Cell-Derived Hepatocytes.

Hepatocyte transplantation has the potential to cure inherited liver diseases, but its application is impeded by a scarcity of donor livers. Therefore, we explored whether transplantation of hepatocyte-like cells (iHeps) differentiated from human induced pluripotent stem cells (iPSCs) could ameliorate inherited liver diseases. iPSCs reprogrammed from human skin fibroblasts were differentiated to iHeps, which were transplanted into livers of uridinediphosphoglucuronate glucuronosyltransferase-1 (UGT1A1)-deficient Gunn rats, a model of Crigler-Najjar syndrome 1 (CN1), where elevated unconjugated bilirubin causes brain injury and death. To promote iHep proliferation, 30% of the recipient liver was X-irradiated before transplantation, and hepatocyte growth factor was expressed. After transplantation, UGT1A1+ iHep clusters constituted 2.5%-7.5% of the preconditioned liver lobe. A decline of serum bilirubin by 30%-60% and biliary excretion of bilirubin glucuronides indicated that transplanted iHeps expressed UGT1A1 activity, a postnatal function of hepatocytes. Therefore, iHeps warrant further exploration as a renewable source of hepatocytes for treating inherited liver diseases.

Functional analysis of a chromosomal deletion associated with myelodysplastic syndromes using isogenic human induced pluripotent stem cells.

Chromosomal deletions associated with human diseases, such as cancer, are common, but synteny issues complicate modeling of these deletions in mice. We use cellular reprogramming and genome engineering to functionally dissect the loss of chromosome 7q (del(7q)), a somatic cytogenetic abnormality present in myelodysplastic syndromes (MDS). We derive del(7q)- and isogenic karyotypically normal induced pluripotent stem cells (iPSCs) from hematopoietic cells of MDS patients and show that the del(7q) iPSCs recapitulate disease-associated phenotypes, including impaired hematopoietic differentiation. These disease phenotypes are rescued by spontaneous dosage correction and can be reproduced in karyotypically normal cells by engineering hemizygosity of defined chr7q segments in a 20-Mb region. We use a phenotype-rescue screen to identify candidate haploinsufficient genes that might mediate the del(7q)- hematopoietic defect. Our approach highlights the utility of human iPSCs both for functional mapping of disease-associated large-scale chromosomal deletions and for discovery of haploinsufficient genes.

The exosome complex establishes a barricade to erythroid maturation.

Complex genetic networks control hematopoietic stem cell differentiation into progenitors that give rise to billions of erythrocytes daily. Previously, we described a role for the master regulator of erythropoiesis, GATA-1, in inducing genes encoding components of the autophagy machinery. In this context, the Forkhead transcription factor, Foxo3, amplified GATA-1-mediated transcriptional activation. To determine the scope of the GATA-1/Foxo3 cooperativity, and to develop functional insights, we analyzed the GATA-1/Foxo3-dependent transcriptome in erythroid cells. GATA-1/Foxo3 repressed expression of Exosc8, a pivotal component of the exosome complex, which mediates RNA surveillance and epigenetic regulation. Strikingly, downregulating Exosc8, or additional exosome complex components, in primary erythroid precursor cells induced erythroid cell maturation. Our results demonstrate a new mode of controlling erythropoiesis in which multiple components of the exosome complex are endogenous suppressors of the erythroid developmental program.

Surface expression of HLA-G is involved in mediating immunomodulatory effects of placenta-derived multipotent cells (PDMCs) towards natural killer lymphocytes.

Interactions between maternal natural killer lymphocytes (NKs) and fetal tissues are important in mediating maternal-fetal tolerance. We therefore investigated the interactions of NKs to placenta-derived multipotent cells (PDMCs) isolated from the term human placenta. PDMCs have similar cell surface marker expression as bone marrow mesenchymal stem cells (BMMSCs) and additionally express human embryonic stem cell markers SSEA-4 and CD-9. Differentiation into the tri-mesodermal lineages of osteoblastic, adipocytic, and chondrogenic phenotypes can be readily achieved under the appropriate conditions. We found that PDMCs are more resistant to NK-mediated lysis than the major histocompatibility complex (MHC) class-I null target cell K562, and can suppress NK secretion of interferon-γ (IFN-γ). Moreover, as third-party cells, PDMCs suppressed the cytotoxic effects of cytokine-stimulated NKs on K562. Pretreatment of PDMCs with IFN-γ, a proinflammatory cytokine, surprisingly enhanced such immunosuppressive effects. Cell-cell contact between NKs and PDMCs is required for suppressive effects, which are partially mediated by slight upregulation of the NK inhibitory receptor killer inhibitory receptor and downregulation of the activating receptor NKp30. Moreover, enhancement of PDMC suppressive effects is also mediated by IFN-γ-induced surface expression of HLA-G--an immunomodulatory nonclassical MHC class I molecule--on PDMCs, as seen by partial reversibility with HLA-G neutralizing antibodies. With its broad immunosuppressive properties, PDMCs may represent a potential cell source for therapeutic use.

Brief report--human embryonic stem cell-derived mesenchymal progenitors possess strong immunosuppressive effects toward natural killer cells as well as T lymphocytes.

The derivation of mesenchymal progenitors from human embryonic stem cells (hESCs) has recently been reported. We studied the immune characteristics of these hESC-derived mesenchymal progenitors (EMPs) and their interactions with T lymphocytes and natural killer cells (NKs), two populations of lymphocytes with important roles in transplantation immunology. EMPs express a number of bone marrow mesenchymal stromal cell (BMMSC) markers, as well as the hESC marker SSEA-4. Immunologically, EMPs do not express HLA-DR or costimulatory molecules. On the other hand, HLA-G, a nonclassic MHC I protein involved in mediating maternal-fetal tolerance, can be found on the surface of EMPs, and its expression is increased after interferon-gamma stimulation. EMPs can suppress CD4(+) or CD8(+) lymphocyte proliferation, similar to BMMSCs. However, EMPs are more resistant to NK-mediated lysis than BMMSCs and can suppress the cytotoxic effects of activated NKs, as well as downregulating the NK-activating receptors NKp30 and NKp46. With their broad immunosuppressive properties, EMPs may represent a new potential cell source for therapeutic use.