IntroductionIn guiding human embryonic stem cell (hESC) technology toward the clinic, 1 key issue to be addressed is a lack of standardization in the culture and maintenance of hESCs. In the absence of mouse embryonic fibroblast (MEF) feeder layers, many researchers rely on "conditioning" in which medium is first exposed to MEFs to acquire soluble factors that support the propagation of undifferentiated hESCs in culture. It has been difficult to discern how MEF conditioning enables hESCs to maintain an undifferentiated state. Other common features of more recently developed hESC culture conditions include the presence of fibroblast growth factor-2 (FGF2), the absence of serum, and the presence of a serum substitute such as KnockOut Serum Replacer (KSR, proprietary formulation; Invitrogen, Carlsbad, CA). [1][2][3] Other factors suggested to play a role in supporting the maintenance of hESCs include transforming growth factor ␤1 (TGF␤1), 4 activin A (ActA), 5,6 platelet-derived growth factor (PDGF) and sphingosine-1-phosphate, 7 BIO, a small-molecule inhibitor of GSK3␤, 8 and neurotrophins. 9 Several defined medium systems have been described for hESCs and are based upon FGF2 in combination with nodal, 10 TGF␤1, GABA, and pipecolic acid, plus lithium chloride, 11 Wnt3a plus April/BAFF, 12 or the N2/B27 supplements. 13 Although these studies have focused on identifying growth factors and conditions that support the proliferation of undifferentiated hESCs, little is known about the cell-surface receptors that are activated when hESCs are exposed to conditions favorable for self-renewal. A number of receptor tyrosine kinases (RTKs) are expressed at high levels on hESCs, 14 including insulin-like growth factor-1 receptor (IGF1R), fibroblast growth factor receptor (FGFR1), and EPHA1, as well as ERBB2 and ERBB3 (which are members of the epidermal growth factor receptor [EGFR] family), while expression of FGFR2 (EGFR) FGFR4, vascular endothelial growth factor receptor-2 (VEGFR2), IGFR2, KIT, and RET has also been detected. 15,16 RTKs are likely to be central signaling effectors 17 that influence survival, apoptosis, proliferation, or differentiation decisions in pluripotent cells. To determine if any of these RTKs are involved in self-renewal, we simultaneously interrogated the tyrosine phosphorylation status of 42 RTKs in hESCs grown in MEF-conditioned medium (CM) and developed a defined medium for hESC culture. The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on May 7, 2018. by guest www.bloodjournal.org From Materials and methods Cell cultureThe National Institutes of Health (NIH)-registered H1, BG01, BG02, and BG03 hESC lines, as well as CyT49, an hESC line isolated using human feeder cells under good manufacturing process (GMP) conditions (Novocell, San Diego, CA), we...

G9a and GLP are conserved protein methyltransferases that play key roles during mammalian development through mono-and dimethylation of histone H3 Lys 9 (H3K9me1/2), modifications associated with transcriptional repression. During embryogenesis, large H3K9me2 chromatin territories arise that have been proposed to reinforce lineage choice by affecting high-order chromatin structure. Here we report that in adult human hematopoietic stem and progenitor cells (HSPCs), H3K9me2 chromatin territories are absent in primitive cells and are formed de novo during lineage commitment. In committed HSPCs, G9a/GLP activity nucleates H3K9me2 marks at CpG islands and other genomic sites within genic regions, which then spread across most genic regions during differentiation. Immunofluorescence assays revealed the emergence of H3K9me2 nuclear speckles in committed HSPCs, consistent with progressive marking. Moreover, gene expression analysis indicated that G9a/GLP activity suppresses promiscuous transcription of lineage-affiliated genes and certain gene clusters, suggestive of regulation of HSPC chromatin structure. Remarkably, HSPCs continuously treated with UNC0638, a G9a/GLP small molecular inhibitor, better retain stem cell-like phenotypes and function during in vitro expansion. These results suggest that G9a/GLP activity promotes progressive H3K9me2 patterning during HSPC lineage specification and that its inhibition delays HSPC lineage commitment. They also inform clinical manipulation of donor-derived HSPCs.

Purpose: We developed a method to monitor copy number variations (CNV) in plasma cell-free DNA (cfDNA) from patients with metastatic squamous non-small cell lung cancer (NSCLC). We aimed to explore the association between tumor-derived cfDNA and clinical outcomes, and sought CNVs that may suggest potential resistance mechanisms. Experimental Design: Sensitivity and specificity of low-pass whole-genome sequencing (LP-WGS) were first determined using cell line DNA and cfDNA. LP-WGS was performed on baseline and longitudinal cfDNA of 152 patients with squamous NSCLC treated with chemotherapy, or in combination with pictilisib, a pan-PI3K inhibitor. cfDNA tumor fraction and detected CNVs were analyzed in association with clinical outcomes. Results: LP-WGS successfully detected CNVs in cfDNA with tumor fraction !10%, which represented approximately 30% of the first-line NSCLC patients in this study. The most frequent CNVs were gains in chromosome 3q, which harbors the PIK3CA and SOX2 oncogenes. The CNV landscape in cfDNA with a high tumor fraction generally matched that of corresponding tumor tissue. Tumor fraction in cfDNA was dynamic during treatment, and increases in tumor fraction and corresponding CNVs could be detected before radiographic progression in 7 of 12 patients. Recurrent CNVs, such as MYC amplification, were enriched in cfDNA from posttreatment samples compared with the baseline, suggesting a potential resistance mechanism to pictilisib. Conclusions: LP-WGS offers an unbiased and highthroughput way to investigate CNVs and tumor fraction in cfDNA of patients with cancer. It may also be valuable for monitoring treatment response, detecting disease progression early, and identifying emergent clones associated with therapeutic resistance.

equity in the company. KNK holds stock in Illumina, Inc. GRO is an employee of Foundation Medicine with equity in Roche. MVS is an employee of, and shareholder in, McKesson Corporation, and is a clinical advisor/serves on the advisory board for GRAIL. EAK is a consultant for GRAIL, Inc. and Cellanyx, LLC. The Mayo Clinic was compensated for MCL's advisory board activities for GRAIL, Inc.Research.

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