To apply massively parallel and clonal sequencing (next generation sequencing or NGS) to the analysis of forensic mixed samples.A duplex polymerase chain reaction (PCR) assay targeting the mitochondrial DNA (mtDNA) hypervariable regions I/II (HVI/HVII) was developed for NGS analysis on the Roche 454 GS Junior instrument. Eight sets of multiplex identifier-tagged 454 fusion primers were used in a combinatorial approach for amplification and deep sequencing of up to 64 samples in parallel.This assay was shown to be highly sensitive for sequencing limited DNA amounts ( ~ 100 mtDNA copies) and analyzing contrived and biological mixtures with low level variants ( ~ 1%) as well as “complex” mixtures (≥3 contributors). PCR artifact “hybrid” sequences generated by jumping PCR or template switching were observed at a low level (<2%) in the analysis of mixed samples but could be eliminated by reducing the PCR cycle number.This study demonstrates the power of NGS technologies targeting the mtDNA HVI/HVII regions for analysis of challenging forensic samples, such as mixtures and specimens with limited DNA. Limited and mixed DNA samples are often encountered in forensic cases and pose both technical and interpretation challenges. The highly polymorphic hypervariable regions I/II (HVI/II) of the mitochondrial genome are often successfully used to analyze limited and/or degraded DNA samples . HoweverThe 454 genome sequencing technology is a scalable, clonal, and highly parallel pyrosequencing system that can be used for de novo sequencing of small whole genomes or direct sequencing of DNA products generated by polymerase chain reaction (PCR). The technology uses emulsion PCR (emPCR) to amplify a single DNA sequence to 10 million identical copies. The “clonal sequencing” aspect of the technology enables separation of individual components of a mixture as well as analysis of highly degraded DNA. The clonal sequencing approach used with the 454 GS technology and other next-generation sequencing (NGS) technologies provides a digital readout of the number of reads or individual sequences allowing for a quantitative determination of the components in a mixture . RecentlThe study was conducted between September 2012 and January 2014 at Children’s Hospital Oakland Research Institute, Genetics Department, and was approved by the Children’s Hospital & Research Center Oakland Institutional Review Board.The highly polymorphic HVI and HVII regions of the mitochondrial genome were enriched from genomic DNA samples using a duplex PCR. Mitochondrial DNA HVI/HVII duplex primer sequences described previously in Gabriel et al  were mod® PCR Buffer II without MgCl2 , 2.4 mM MgCl2, 0.2 mM each dNTP , 0.25 U/µL EagleTaq DNA Polymerase , and 0.3 µM each primer. Both HVI (15975-16501) and HVII (15-410) regions were amplified in a single tube using a set of MID-tagged fusion primers. All reactions were performed in GeneAmp® PCR Systems 9600 thermal cycler  using the following cycling conditions: a 14-minute 94°C activation cycle; 34 cycles of amplification at 94°C for 15 seconds, 65°C for 30 seconds, and 72°C for 30 seconds; and a final 10-minute 72°C extension cycle. PCR products were analyzed on 1.5% agarose gels in 1X Tris-Acetate-EDTA electrophoresis buffer (TAE) stained with 0.5 µg/mL ethidium bromide to confirm successful amplification of the target regions and to assess primer dimer and PCR specificity.Each sample was amplified in a 50 µL PCR reaction containing 1.2X GeneAmp® dsDNA Assay kit (Life Technologies) to determine the DNA amount in copy number for sample normalization and subsequent library pooling. An approximately equal copy number of each amplified sample was combined into a single library pool. Small fragment DNA or primer dimers were removed from the pooled library using Agencourt AMPure XP  with 1:1 AMPure to DNA sample volumetric ratio. The Agilent DNA 1000 kit  was used following manufacturer’s protocol in order to compare the library pool before and after AMPure purification to confirm the removal of primer dimers. The concentration of the purified library pool was estimated using Quant-iT PicoGreen® dsDNA kit (Life Technologies) or Library Quantification Kit - 454 FLX  to estimate the DNA copy number of the library pool.Each PCR product was quantified using Quant-iT PicoGreen5 molecules/uL in order to add a total of 10 uL to achieve a 0.4:1 DNA to bead ratio in emPCR. The optimal DNA to bead ratio was determined to be 0.4:1 based on the number of high quality pass filter reads. Targeting the optimal DNA:bead ratio is critical to avoid the incorporation of more than one molecule of DNA per bead or insufficient bead recovery. The finalized library pool was amplified using the GS Junior Titanium emPCR Kit (Lib-A) (Roche Life Sciences) following the manufacturer’s protocol. All successfully prepared libraries were sequenced on the 454 GS Junior System . Amplicon Variant Analyzer (AVA) was used to analyze the 454 sequence data and identify SNP variants compared to the revised Cambridge sequence (rCRS)  to the concentration of 4 × 10e (rCRS) .Sensitivity and concordance studies. A sensitivity study was conducted to determine the limits of the assay as follows. The mtDNA copy number and nDNA concentration of a control DNA sample K562 High Molecular Weight,  was quantified using a mt:nu qPCR assay and serially diluted (® dsDNA HS Assay Kit (Life Technologies), combined into a single library pool, and sequenced using the 454 GS Jr. To determine the accuracy of the assay, 454 HVI/HVII sequence data were generated and analyzed from previously collected blood derived DNA samples from four population groups  ( diluted . Thirtee diluted . The 13 apanese) . Sanger apanese) .Contrived and biological mixture studies. Mixture studies (two and three person mixtures) were conducted to determine the sensitivity of the assay for detecting and resolving the minor sequences in a mixed specimen. Two Coriell Cell line DNA samples , differing at 10 base positions (7 in HVII and 3 in HVI) determined previously using Sanger sequencing were mixed together at various ratios based on the mtDNA copy number. The mtDNA copy number was determined using a mt:nu qPCR assay described by Timken et al . The sequences generated by the 454 HVI/HVII NGS assay showed concordance with the Sanger sequencing results at all base substitution positions (point mutations) when a 10% threshold was applied to the data analyzed using the 454 AVA software. A 10% threshold was applied to account for the lower sensitivity of Sanger sequencing for minor base detection (10%-15%) compared to 454 sequencing (1%-3%). Minor base differences were observed at some base positions at levels below the 10% threshold with 454 sequencing. These minor base differences observed at very low frequency (<1%) can be attributed to PCR or sequencing error or heteroplasmy; further analysis is needed in order to distinguish true signal from noise.As expected, 454 pyro-sequencing errors were observed as insertion/deletions (IN/DEL) above the 10% threshold within the sequence surrounding the two homopolymer C stretches in the HVI/HVII regions, which resulted in sequence alignment errors using the AVA software. Typically, these IN/DEL sequencing errors were observed in only a single direction and/or the base calls for a given position were imbalanced. Specifically, pyro-sequencing errors and/or length heteroplasmy in the HVI region spanning base positions 16180-16195 (5′-AAAACCCCCTCCCCAT-3′) resulted in alignment issues using the AVA software. An A/C mutation at position 16183 and any C/T transition observed between base positions 16184 to 16193 of this region was reported as a combination of IN/DEL of A/C or C/T, with imbalanced frequencies between read directions. The SNPs in the homopolymer region were detected, but were not reported correctly due to alignment issues from homopolymer pyro-sequencing errors or potentially length heteroplasmy using the 454 AVA software.Additionally, pyro-sequencing errors and/or length heteroplasmy within the homopolymer region surrounding the C-stretch in the HVII region spanning positions 286- 315 (5′-AAAAAATTTCCACCAAACCCCCCCTCCCCC-3′) were observed. Within this region, an A deletion was sometimes reported at base position 291, and position 295 was reported as a mixture of C/T followed by C deletion both with directionally imbalanced frequencies using 454 AVA software. These observations can be partially explained as a sequence alignment issue for the specific regions with homopolymers within the HVI/HVII sequences using the 454 sequencing platform as it is well established that pyro-sequencing has a relatively high error rate in homopolymer regions .We conducted a two person mixture study to determine the sensitivity of the 454 HVI/HVII NGS assay for detecting minor components in a mixture. 454 HVI/HVII sequencing results are presented in in vitro crossover artifact) was observed in each of the complex mixtures as chimeric or hybrid sequences were present at low levels (A complex mixture study (≥3 contributors) was conducted to test the 454 HVI/HVII assay’s capability to detect and resolve multiple sequences in a mixed sample. 454 HVII NGS results for a three person mixture are presented in w levels . A totalJumping PCR, a phenomenon where a single strand of DNA partially extended on one template can act as a primer on the other template to generate an amplicon with chimeric sequences, can occur during late PCR cycles when the number of amplicon copies is very high and the concentration of primers and enzyme is low . As expeTo further determine the detection limits of the 454 HVI/HVII NGS assay, a heteroplasmy study was conducted. HV+ HaploArray  and 454 HVI/HVII NGS results from five hairs, blood, and buccal samples from one twin pair with heteroplasmy at position 16093 are presented in We developed a duplex PCR assay targeting the mtDNA (HVI/HVII) using eight sets of 454 MID-tagged fusion primers in a combinatorial approach for deep sequencing 64 samples in parallel on a 454 GS Jr. Compared with Sanger sequencing of the same samples, the sequence concordance was 100% outside of the homopolymeric regions in HVI and HVII using a 10% cut-off for the 454 sequencing data. Sequencing errors appearing as IN/DEL mutations in homopolymer regions were observed. This issue is a well known feature of NGS based on pyrosequencing . Recent Our assay was shown to be highly sensitive for sequencing limited DNA amounts ( ~ 100 mtDNA copies) and detecting mixtures with low level variants ( ~ 1%) as well as “complex” mixtures (≥3 contributors) not possible to analyze by Sanger sequencing. The minor base was reliably detected at each of the mixed base positions and the observed frequencies were similar to the expected frequencies using 454 sequencing with ~ 600-1000 reads per amplicon. Further, we characterized jumping PCR and resulting hybrid artifact sequences that can arise in amplification of mixed samples and noted that the frequency of the phenomenon was template amount- and cycle-dependent. This effect, which occurs at late cycles of PCR, can be decreased by simply lowering the amplification cycle number. In addition, such chimeric PCR artifacts can be potentially filtered through bioinformatics tools.The clonal nature of NGS provides a powerful and quantitative means of deconvoluting mixtures, a particularly challenging category of forensics specimens. Multiple contributors to a mixture can be detected by analyzing the different clonal sequence reads identified in the sequence analysis and their contribution quantified by simply counting the number of sequence reads. This digital analysis is much more precise than estimating peak height or area for STR markers or analyzing Sanger electropherograms for mtDNA sequences. For chromosomal markers, one must make assumptions about which alleles in a mixture “go together” as a genotype. Many statistical approaches to mixture analysis consider all possible genotype combinations and, in some cases, different numbers of contributors. Mitochondrial DNA, on the other hand, is a particularly useful marker for deconvoluting mixtures because, potential heteroplasmy aside, each distinct mtDNA sequence corresponds to (at least) one contributor. Statistical analyses can capture and incorporate the probability that one mtDNA sequence could correspond to more than one contributor. Sequencing the entire mtDNA genome rather than the HVI/II regions reported here will provide additional valuable discrimination power and several groups have recently reported NGS analyses using long range PCR -31. Rece


Throughout life adult animals crucially depend on stem cell populations to maintain and repair their tissues to ensure life-long organ function. Stem cells are characterized by their capacity to extensively self-renew and give rise to one or more differentiated cell types. These powerful stem cell properties are key to meet the changing demand for tissue replacement during normal lung homeostasis and regeneration after lung injury. Great strides have been made over the last few years to identify and characterize lung epithelial stem cells as well as their lineage relationships. Unfortunately, knowledge on what regulates the behavior and fate specification of lung epithelial stem cells is still limited, but involves communication with their microenvironment or niche, a local tissue environment that hosts and influences the behaviors or characteristics of stem cells and that comprises other cell types and extracellular matrix. As such, an intimate and dynamic epithelial-mesenchymal cross-talk, which is also essential during lung development, is required for normal homeostasis and to mount an appropriate regenerative response after lung injury. Fibroblast growth factor 10 (Fgf10) signaling in particular seems to be a well-conserved signaling pathway governing epithelial-mesenchymal interactions during lung development as well as between different adult lung epithelial stem cells and their niches. On the other hand, disruption of these reciprocal interactions leads to a dysfunctional epithelial stem cell-niche unit, which may culminate in chronic lung diseases such as chronic obstructive pulmonary disease (COPD), chronic asthma and idiopathic pulmonary fibrosis (IPF). The adult lung epithelium is replaced over time, albeit very infrequently in comparison to organs exhibiting constant cellular turnover such as the skin and intestine. However, after injury, the lung harbors a remarkable capacity to regenerate and restore its function. This is dramatically illustrated after unilateral pneumectomy, which induces an expansion of stem cell populations and compensatory growth of the remaining lung to re-establish respiratory capacity . The comLineage tracing experiments during normal homeostasis have identified three main stem cell populations responsible for maintaining the lung epithelium: basal cells, club cells and ATII cells. Their lineage relationships are depicted in Figure 2 or naphthalene injury kills most luminal cell types leaving behind a few surviving club cells and an intact basal cell layer  ducts has been shown to drive epithelial regeneration after severe epithelial injury using a tracheal transplant model of hypoxic ischemia, which destroys nearly all epithelial cells  did not reveal any contribution of BASCs to alveolar regeneration [Scgb1a1CreER mice however, it was shown that Scgb1a1+ club cells in the distal airway can give rise to both ATI and ATII cells following catastrophic bleomycin- or H1N1-mediated injury [A widely used model to study epithelial regeneration of the bronchioles is naphthalene injury. In the bronchioles, naphthalene selectively depletes club cells except for the few naphthalene-resistant club cells  located near NEBs ,17 and al injury ,22  α6+/β4+ alveolar epithelial stem cell population has recently been characterized, having the potential to give rise to both ATII and club cells in vitro and in vivo after injury [Importantly, although BASCs/DASCs can contribute to alveolar regeneration, ATII cells are the main stem cell population in the lung respiratory epithelium involved in replenishing ATII cells after their diphtheria toxin-mediated depletion , re-estar injury ,28 , which is expressed in the distal mesenchyme and is regulated by Wnt signaling [Sox9 and repressing Sox2 expression [Fgf10 expression and presentation via HS proteoglycans is necessary during early development so that high levels are achieved distally to promote rapid expansion of embryonic progenitors. Cell surface-tethered HS chains play pivotal roles in the local retention of Fgf ligands and can spread Fgf signaling to adjacent cells within a short range [Fgf10 suppression around the developing airway, as well as during late gestation and postnatal development, is crucial to allow for proper maturation of the lung epithelium [Fgf10 overexpression at later stages of lung development prevents the alveolar differentiation program through the induction of Sox9 expression [Fgf10-expressing cells in the distal mesenchyme during early lung development are progenitors for airway smooth muscle cells (ASMCs) [During lung development, a complex interplay between endoderm and mesoderm defines early developmental competence and cell fate. This cross-talk is orchestrated by key signals including Fgfs, Wnts, retinoic acid, Shh and Tgfβ (reviewed in ). Proximignaling -36, actspression ,38  ,48,49 (F (ASMCs) .Fgf10 is also expressed in between tracheal cartilage rings during lung development [Fgf10 knockout tracheas show a 50% reduction in basal cells compared to wild type tracheas [Fgf10 during lung development results in the ectopic differentiation of a large subset of Sox2+ airway epithelial cells into the basal cell lineage all along the conducting airway. Interestingly, inhibition of Fgf signaling from E15.5 until E18.5 resulted in a failure to maintain basal cells in the mouse trachea [elopment -53. In telopment . Fgf10 ktracheas . In addi trachea .Much progress has been made in identifying the different stem cell populations involved in the maintenance and repair of the adult lung epithelium as well as their lineage relationships. However, much less is known about the nature of stem cell niches in the lung and how they influence stem cell behavior.Fgf10 expression [Fgf10 overexpression enhances airway epithelial regeneration, whereas inhibition of Fgf10 signaling reduces regenerative capacity after naphthalene injury [Fgf10 by ASMCs is observed after different types of airway epithelial injury, including ozone and bleomycin injury [Cellular and molecular mechanisms important during development are often reactivated during adult injury repair and in disease. As such, we have recently identified ASMCs as a niche for club stem cells . After npression ,55, whice injury ,56, unden injury .Fgf10 expression is the highest [In the mouse trachea, label-retaining basal stem cells preferentially reside in SMG ducts and in intercartilage regions ,58, two  highest -53,59. W highest  remains Interestingly, direct cell-cell contact between basal cells and club stem cells in the tracheal epithelium inhibits the reprogramming of club stem cells into basal stem cells . In this− club cells, characterized by expression of N1ICD, Scgb3a2, Uroplakin 3a (Upk3a) and SSEA-1, has been identified juxtaposed to Ascl1-expressing NEBs and remain largely uncommitted during development but can give rise to Scgb1a1+ club and ciliated cells in the adult lung [Ascl1−/−mice suggesting that the induction and/or maintenance of these club-like precursor cells is highly dependent on the presence of NEBs in the developing bronchi [A novel subpopulation of Scgb1a1ult lung ,61. This bronchi ,61. Thes bronchi ,62 led t bronchi . TherefoFgf10[Sftpc[− alveolar stem cells enriched for α6/β4 integrins relies on co-culture with Sca1+ mesenchymal cells, which can be substituted by exogenous Fgf10 [− and Fgf10+ adult lung mesenchymal stromal cells (MSC) exists, which functions as a progenitor for LIFs and can support lung epithelial stem cell growth in vitro due to its Fgf10 expression. As during lung development, Fgf10 expression in these cells can be inhibited by Tgfβ signaling [A similar cross-talk between epithelial stem cells and their niche is essential to maintain and regenerate the distal respiratory epithelium -65. In tFgf10,54, whicf10[Sftpc,72,73. Af10[Sftpc. Similarus Fgf10 . An addiignaling ,74,75.(Tsp1). This endothelial-derived Tsp1 then drives the differentiation of BASCs into the alveolar lineage via a feedback loop [Bmp4 and Tsp1 expression is downregulated, which was shown to favor the differentiation of BASCs into the club cell lineage [Bmp4 is a main target gene of Fgf10 signaling [Fgf10 expression is upregulated in ASMCs and Fgf10 signaling in BASCs is activated after both naphthalene and bleomycin injury [Bmp4 secreted by BASCs after bleomycin injury acts on endothelial cells via Bmpr1a to trigger calcineurin/NFATc1-dependent expression of thrombospondin-1 ack loop . Interes lineage . Consideignaling ,76-78, in injury  , are characterized by the progressive remodeling of the airways and/or parenchyma, which irreversibly leads to lung dysfunction. In most cases their etiology is not well understood and current therapeutics are aimed at ameliorating rather than curing these diseases. Tissue architecture remodeling involves pathological changes in the composition and physiological function of the epithelium. During normal homeostasis as well as after injury, lung epithelial stem cells need to give rise to the right amount of differentiated progeny while maintaining their self-renewing ability depending on the physiological context. Disrupting this balance can lead to excessive differentiation and stem cell exhaustion, hypoplasia or squamous metaplasia. Such abnormal changes in stem cell function may result from inherent (epi)genetic changes or a dysfunctional stem cell niche. Furthermore, pathological changes to the lung epithelium may drastically influence the crosstalk between stem cells and their niche, which further contributes to disease progression by inducing abnormal changes in mesenchymal components, such as ASMC hyperplasia and fibroblast activation leading to excessive ECM deposition and fibrotic scarring. Chronic injury and prolonged activation of repair pathways are thought to result in decreased repair potential by exhausting the stem cell pool, which leads to defective repair and progressive airway remodeling by promoting a fibrotic response .Pathological hallmarks in asthma are an increase in smooth muscle mass surrounding the airway wall, SMG hyperplasia and thickening of the basal lamina beneath the seemingly ‘normal’ BM . The latFlg gene) lead to a defect in epithelial barrier function resulting in increased epithelial permeability and penetration of exogenous substances [New paradigms in asthma research point to a central role for the epithelium and chronic activation of epithelial repair pathways as a major cause of airway remodeling and the development of persistent alterations in airway function . This isbstances ,132. Disbstances -135. Thebstances ,136,137.bstances .Snail1 expression is induced downstream of Fgf10 or Notch signaling in club stem cells during airway epithelial regeneration resulting in a transient EMT [Snail1 knockout showed that Snail1 plays a crucial role in the progression of liver fibrosis without driving a full EMT. Instead, Snail1 was shown to drive the expression of growth factors, ECM components and pro-inflammatory mediators [Interestingly, the Wnt pathway is induced in ASMCs in a mouse model for asthma , whereasient EMT . Interesediators .IPF is a devastating, age-related lung disease with an unknown etiology that is refractory to treatment and has a poor survival rate. Widespread damage to the epithelium and/or exhaustion of the epithelial stem cell pool, such as in IPF patients with certain telomerase mutations -149, ult+ basal cells, which are rare in the normal epithelium [Fgf10 haploinsufficiency is linked to emphysema in COPD in humans, which further emphasizes the significance of Fgf10 expression in adult lung stem cell niches and its role in homeostasis and regeneration [COPD is another chronic lung disorder that develops mainly in smokers and clinically manifests itself through chronic bronchitis and emphysema. Epithelial remodeling includes basal cell hyperplasia and squamous metaplasia with several layers of Krt14ithelium . In addiithelium . Interesneration .in vitro in organoid cultures will be crucial for long-term expansion of lung stem cells and the development of cell replacement therapies. As such, the feasibility of colon stem cell therapy based on transplantation of colon organoids in a damaged mouse colon has recently been demonstrated [Stem cells are indispensible during normal homeostasis and regeneration to restore the form and function of an organ after injury. Great strides are being made toward identifying and characterizing stem cell populations in the adult lung. It is, however, important to keep in mind that stem cells do not act independently to perform these functions. Stem cells are subjected to tight regulatory processes so that they are activated and give rise to the right number and type of differentiated progeny at the appropriate time and place in a given biological context. In that regard, stem cells must be mobilized quickly in response to injury, but once the tissue has been restored, stem cells must also be able to revert back to their quiescent state. How exactly this information is communicated to stem cells is not well understood, but may involve dynamic feedback mechanisms between stem cells and their niche. We are now only beginning to understand the cellular and molecular constituents of the stem cell niches in the lung and how stem cell behavior is influenced by the wide variety of external signals provided by their niche. In this review, we have advocated the involvement of Fgf10, which is expressed in several stem cell niches in the lung, in stem cell maintenance and activation after injury. Developmental pathways are often recapitulated during lung repair and their chronic and sustained activation may lead to lung remodeling observed in chronic diseases such as IPF, COPD and chronic asthma. As such, determining the regulatory pathways involved in stem cell-niche interactions during adult lung homeostasis and repair after injury will pave the way for a better understanding of the molecular mechanisms underlying these devastating disorders for which there are currently few or inefficient treatments. Moreover, effectively recreating the stem cell niche nstrated .ASMC: airway smooth muscle cell; ATI: alveolar type I; ATII: alveolar type II; BADJ: bronchioalveolar duct junction; BASC: bronchioalveolar stem cell; BM: basement membrane; COPD: chronic obstructive pulmonary disease; DASC: distal airway stem cell; EGF: epidermal growth factor; ECM: extracellular matrix; EMT: epithelial-mesenchymal transition; FGF10: fibroblast growth factor 10; FN: fibronectin; GCM: goblet cell metaplasia; HS: heparin sulfate; IPF: idiopathic pulmonary fibrosis; Itg: integrin; Krt: cytokeratin; LIF: lipofibroblast; NE: neuroendocrine; NEB: neuroendocrine body; SMG: submucosal gland; Th2: T-helper 2; Trp63 or p63: transformation-related protein 63; Tsp1: thrombospondin-1; Upk3a: uroplakin 3a.The authors declare that they have no competing interests.TV and SDL developed the review concept, performed the literature review, created figures, wrote and revised the manuscript. Both authors read and approved the final version of the manuscript.TV is a predoctoral candidate. SDL is an associate professor of pediatrics.


Pluripotent stem cells represent a promising source of differentiated tissue-specific stem and multipotent progenitor cells for regenerative medicine and drug testing. The realisation of this potential relies on the establishment of robust and reproducible protocols of differentiation. Several reports have highlighted the importance of biomaterials in assisting directed differentiation. Graphene oxide (GO) is a novel material that has attracted increasing interest in the field of biomedicine. In this study, we demonstrate that GO coated substrates significantly enhance the differentiation of mouse embryonic stem (ES) cells to both primitive and definitive haematopoietic cells. GO does not affect cell proliferation or survival of differentiated cells but rather enhances the transition of haemangioblasts to haemogenic endothelial cells, a key step during haematopoietic specification. Importantly, GO also improves, in addition to murine, human ES cell differentiation to blood cells. Taken together, our study reveals a positive role for GO in haematopoietic differentiation and suggests that further functionalization of GO could represent a valid strategy for the generation of large numbers of functional blood cells. Producing these cells would accelerate haematopoietic drug toxicity testing and treatment of patients with blood disorders or malignancies. Bone marrow transplantations are well-established cellular therapies for the treatment of a variety of malignant or genetic disorders of blood cells246in vitro ESC-derived haematopoietic lineage specification is initiated with a mesodermal-derived precursor termed the blast colony forming cell (BL-CFC), which is the in vitro equivalent of the haemangioblast, a mesodermal progenitor with both endothelial and haematopoietic potential810121315The in vivo development would be essential to generate fully differentiated and functional cells18in vivo architecture and microenvironment provide novel opportunities for the directed differentiation of PSCs to desired lineage202324Small molecule modulators of signalling pathways and epigenetic modifiers can exert profound effect on the maintenance and differentiation of ESCsGraphene and related materials have been shown to be excellent supporting scaffolds for PSC differentiation due to their mechanical stability and physiochemical properties28303435In this study, we investigated the relevance of GO in ESC-derived haematopoietic development and established that GO significantly enhances the generation of haematopoietic progenitor cells from both murine and human ESCs. More specifically we determined that GO enhances the transition of haemangioblast to haemogenic endothelium and thereby induces both primitive as well as definitive haematopoiesis.The GO used in this study was comprised of 100% monolayer flakes (~1 nm thick) and 5–30 μm in lateral size, as determined by atomic force microscopy (AFM) and scanning electron microscopy (SEM). The excellent distribution of 100% monolayer GO flakes which we have obtained in our oxidation and exfoliation procedure is evidenced by the uniform 1 layer step-height in AFM as well as the uniform secondary electron contrast of the flakes seen in SEM. In order to obtain a statistically significant sampling, we combined random SEM and AFM imaging and analysis to cover a large number of flakes with SEM secondary electron contrast, and to validate the SEM contrast using AFM analysis, which is a more direct measure of thickness but can only cover smaller regions in each scan. The C/O ratio in the GO was ~1 as determined by X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy D/G peak intensity ratio was ~0.9. GO coated glass coverslips were used for this study; the GO coating had 100% surface coverage and thickness varied from 10 to 15 nm, indicating few-layered uniform GO deposition .The role of GO and graphene on the early differentiation of ESCs to different germ layers has been previously reported89+ cells generated in the presence of GO was either due to an enhanced production of these cells by the haemangioblasts or to altered cell cycle status and/or survival of these cells in these culture conditions. EdU incorporation assay was used to assess cell proliferation and cell cycle progression of the haemangioblast cultures. These analyses did not reveal any significant changes in EdU labelling indicating that haemangioblasts cultures on GO and GE proliferate at similar rate at day 1 . Cultures previously maintained on GO generated a higher number of haematopoietic colonies at all time points examined  in which the green fluorescent protein (GFP) encoding gene has been knocked-in the embryonic βH1 haemoglobin locus, which is specifically transcribed in primitive erythroid cells. As shown in ytometry . On averytometry . Togethe+ cells that are negative for GFP and therefore correspond to definitive haematopoietic cells. Again the frequency of GFP−CD41+ cells was significantly higher when cultured on GO  cell population was sorted from 2-day haemangioblasts cultured on GE and seeded on either GE or GO-coated cover slips. The transition to the haematopoietic lineage was monitored by FACS for gain of CD41 expression and loss of TIE2 expression. As shown in Blood progenitors emerge from the HE through a process known as endothelial to haematopoietic transition (EHT)+/c-KIT+ (HE I) cells from FLK1+ haemangioblast cell population cultured on GO or GE (+/c-KIT+ cells when cultured on GO (data not shown). Taken together our results show that GO promotes the transition of haemangioblasts to haemogenic endothelium and thereby positively influence both primitive as well as definitive haematopoiesis , the human equivalent of murine FLK1. The human haemangioblasts were either cultured on GO or control fibronectin (Fib) . In human vitro differentiation. We further demonstrate that this increase in blood cells production stems from a higher rate of transition from murine haemangioblasts to haemogenic endothelium. Significantly, we showed that GO similarly promotes the transition of hESC derived haemangioblasts to haemogenic endothelial cells.In this manuscript, we evaluated the potential influence of GO-coated cell culture substrate on the differentiation of murine ESCs to haematopoietic progenitor cells. Our results indicate that cultures on GO-coated plates result in a significant increase in the production of primitive and definitive haematopoietic cells upon iGO presents several physiochemical properties such as ultra-large surface areas, abundant hydrophilic groups allowing bio-functionalization, and outstanding water solubility that makes it a promising biomaterial in a plethora of application in biotechnology such as drug carriers, biosensors, and antibacterial agents. Our study complements previous works that indicated a beneficial effect of using GO-based biomaterials in the differentiation of pluripotent, mesenchymal and neuronal stem cells333536n vitro recapitulation of complexes environments such as the haematopoietic stem cells (HSC) niche will probably require the identification and combination of several biomaterials that mimic the complex microenvironment in which HSCs reside. Along this line, Liesten et al. have shown that a 3D environment containing collagen and bone marrow derived MSCs can promote expansion of haematopoietic progenitor cellsex vivo upon co-culture with endothelial cellsIncreasing evidence indicates that there is a critical need for micro environmental optimization for efficient maintenance and differentiation of both adult and embryonic stem cells48Our study demonstrates a beneficial effect of GO cell culture substratum on haematopoietic lineage specification in ESC differentiation system. Our results indicate that this positive effect does not result from an increase in EHT or cell proliferation, but is due to the ability of GO to promote haemangioblast to HE transition. Furthermore our results also suggest that substrate dependency varies between different stages of stem cell differentiation to blood. Importantly, similar effects were observed with hESC derived haemangioblasts . The proGraphite oxide was prepared according to a modified Hummers methodMurine embryonic stem cells were differentiated to haemangioblasts as described previouslyMan-5 is a research grade hESC line derived by the UK North West Embryonic Stem Cell Centre®-34 SFM media (Thermo Fisher) supplemented with 0.5% penicillin/streptomycin, L-glutamine (2 mM), ascorbic acid (1 mM), monothioglycerol , transferrin (150 mg/ml) and BMP-4 (10 ng/ml), seeded into low attachment dishes (Corning) to generate embryoid bodies under hypoxic conditions for 4 days. Human haemangioblasts were FACS sorted based on KDR expression and further cultured on fibronectin or GO coated cover slips in serum free StemPro®-34 SFM media (Thermo Fisher) supplemented with 0.5% penicillin/streptomycin, L-glutamine (2 mM), ascorbic acid (1 mM), monothioglycerol , 150 mg/ml transferrin, 10 ng/ml VEGF, 5ng/ml bFGF, 50 ng/ml hSCF, and hIL6 (10 ng/ml) (PropsecBio).Nearly confluent hESCs, maintained in feeder free conditions, were treated with 50 mM EDTA/PBS solution for 1 min at 37 °C and upon addition of defined medium, the cells were manually cut with StemPro EZ Passaging Tool (Life Technologies) to obtain similar size pieces of colonies to generate homogenous sizes of self-aggregated EBs. Cell clumps collected with serological pipette were left to settle down at room temperature and supernatant was removed. Aggregates were resuspended in serum free StemProCells harvested at indicated times were stained with following antibodies: mFLK1-APC (1560), hKDR-Alexafluor 647 (Biolegend-338909), hCD31-APC Cy7 (WM-59), hCD34-FITC (AC 136), CD41-PE-Cy7 (MWReg30), mc-KIT-APCeFluor780 (2B8), mTIE2-PE (eBio12-5987), mCDH5–APC (eBioBV13), mCD45-PerCp5.5 . Stainings were done in 50-ul-reaction volume for 30 minutes on ice. Compensations were performed with beads controls. Acquisition was done on LSRII or Fortessa (BD Biosciences) and data was analysed using FlowJo software (Treestar). Cell sortings were performed on AriaII or AriaIII or Influx sorters (BD Biosciences).Haematopoietic colony forming potentials of cells were assessed in semi solid methylcellulose cultures. 10,000 cells at indicated time points were plated into haematopoietic media containing 1% methylcellulose. The number of primitive erythroid colonies was scored after 4 days of incubation at 37 °C and 5% CO2. Granulocytic and macrophagic colonies were scored after 7 days of incubation.Total RNAs were isolated using QIAGEN RNA Prep KIT (Qiagen). cDNAs were generated using Omni Script (Qiagen) according to manufacturer’s protocols. Taqman probes were used to detect relative levels of indicated genes.Cell cycle analyses were performed using the Click-iT Edu flowcytometry assay kit as per manufacturer’s guidelines (Invitrogen). Apoptosis assays were performed using PE-AnnexinV apoptosis detection kit as per manufacturer’s guidelines (BD Pharmigen).How to cite this article: Garcia-Alegria, E. et al. Graphene Oxide promotes embryonic stem cell differentiation to haematopoietic lineage. Sci. Rep.6, 25917; doi: 10.1038/srep25917 (2016).


Parus major) that breeds at high elevations and winters at lower elevations in the eastern Himalayas. Comparison of population genomics of high altitudinal great tits and those living in lowlands revealed an accelerated genetic selection for carbohydrate energy metabolism  and hypoxia response  in the high altitudinal population. The PI3K-akt, mTOR and MAPK pathways modulate the hypoxia-inducible factors, HIF-1α and VEGF protein expression thus indirectly regulate hypoxia induced angiogenesis, erythropoiesis and vasodilatation. The strategies observed in high altitudinal great tits differ from those described in a closely related species on the Tibetan Plateau, the sedentary ground tit (Parus humilis). This species has enhanced selection in lipid-specific metabolic pathways and hypoxia-inducible factor pathway (HIF-1). Comparative population genomics also revealed selection for larger body size in high altitudinal great tits.Species that undertake altitudinal migrations are exposed to a considerable seasonal variation in oxygen levels and temperature. How they cope with this was studied in a population of great tit ( Cold temperature and low oxygen level present severe challenges for organisms living at high elevations. The strong selection pressures posed by these harsh conditions have resulted in the evolution of key adaptations for hypoxia resistance, cold tolerance and enhanced metabolic capacity in many mammals and birds1234236Parus major) occupies forest habitats all over the Eurasian continent81011Parus humilis)The great tit  values compared to those of the genomic background  to validate SNPs associations with altitude distribution. Between 16 and 32 additional great tit individuals were Sanger sequenced and it was confirmed that the observed amid acid substitutions were nearly fixed in the eastern Himalayas great tits and strongly correlated with their high altitude distribution  to detect signatures of selection associated with high altitude. In the eastern Himalayas group the screen for selective sweep detected 183 genes  under seckground . A detairibution .We also identified signals of selection in developmental genes related to morphogenesis  among great tits in the eastern Himalayas, Central/East and Mongolia. Congruent with this result, we observed that the three groups of great tits differed in their average size: the Mongolian great tits had the largest body size, tail, tarsus and bill lengths, the eastern Himalayas great tits were of intermediate sizes and the Central/East great tits were the smallest see .Parus monticolus), black-throated bushtit , grey-cheeked fulvetta (Alcippe morrisonia) and red-headed tree babbler (Stachyridopsis ruficeps)in situ diversification2021The ecological stratification and environmental heterogeneity of many mountain regions have greatly promoted speciation and diversification in birds and other organismsThe great tits in the eastern Himalayas have a long independent evolutionary history after they diverges from the lowland populations. Since their isolation, they have been exposed to different forces of selection in their local montane environment compared to their lowland ancestors. Our results show that the great tits in the eastern Himalayas cope with their high altitude living condition by evolving adaptations to increase energy metabolism and hypoxia response. Although these adaptations are similar to those reported in the closely related ground titThe great tits in the eastern Himalayas show strong positive selection in genes related to hypoxia response. While most genomic studies on sedentary species  on the Tibetan Plateau have showed evidence of positive selection on genes involved in the HIF-1 and VEGF pathways123424252625Selection in genes involved in the development is also found to be strong in the great tits in the eastern Himalayas compared to those in Central/East China. This may be explained by, on average, the larger body size, longer wing and tarsus lengths in great tits living at high altitude. The larger size and heavier body mass are probably advantageous in heat retention. Furthermore, body size is often correlated with hemoglobin O2 affinity, as larger animals tend to have higher O2 affinity30Tissue samples were obtained from 32 great tits from 13 localities, including 11 individuals from the eastern Himalayas, 11 from Central/East China and 10 from Inner Mongolia and Mongolia . The eas11Novogene Bioinformatics Institute . DNA libraries (500 bp) were constructed according to the manufacturer’s introductions (Illumina). Using a whole genome shotgun strategy we generated a total of 197.84 Gb of paired-end reads of 100 bp length.Genomic DNA was extracted from muscle samples. All samples were sequenced on the Illumina sequencing platform (HiSeq 2000) in To avoid reads with artificial bias in the process of library construction and sequencing , we carried out quality control and filtered out sequences according to the following criteria:(a) any reads with ≥10% unidentified nucleotides (N);(b) any reads with >10 nt aligned to the adapter sequence, allowing ≤10% mismatches;(c) any reads with >50% bases having phred quality <5;(d) putative PCR duplicates generated by PCR amplification in the library construction process (i.e. two paired-end reads were the same).A total of 186.32 Gb  high quality paired-end reads was retained for the further analyses .After quality controls the reads were mapped to the ground tit genome using BWAhttp://evolution.Genetics.Washington.edu/phylip.html). We performed a principal component analysis (PCA) using the package GCTATo carry out population structure and selection analyses, we removed all SNPs with a minor frequency (MAF) < = 0.1 and kept only SNPs that occurred in more than 90% of the individuals. We used all high quality SNPs to infer population structure. To estimate phylogenetic relationships the pairwise genetic distances were calculated among all samples to generate a neighbor-joining (NJ) tree using PHYLIP 3.695 . The generation time for the great tit was set at 1.5 yearsThe demographic history of the three great tit groups was inferred by a pairwise sequentially Markovian coalescence (PSMC) model. As all samples were sequenced at 5x depth, we used all individuals in each group to infer population demographic history to get a consistent result. Parameters were set as ‘−N30 −t15 −r5 −p 45*2’. The mutation rate was set to be 2.78 × 10We reconstructed the palaeodistributions and current distributions for great tits in the eastern Himalayas, Central/East China and Mongolia. Ecological niche models predicted the current distribution in MAXENT43FST statistic  and θπ ratio  values were identified as highly divergent.To detect signatures of selection associated with high altitudes, we measured genome-wide variation between the highland  and lowland groups . We applied a sliding window approach (100-kb window sliding with a step size of 10-kb) to identify selective regions associated with high altitude. Genomic regions with significant high P < 0.05We annotated genes in selected genomic regions using the ground tit genome and a total of 183 genes were identified to be under positive selection in eastern Himalayas’ great tits. These genes were submitted to Gene Ontology and KEGG databases for enrichment analyses. A false discovery rate (FDR) corrected binomial distribution probability approach was used to test significant enriched gene function at a level of FST, θπ and Tajima’s D between high altitude and lowland great tits  to calculate eat tits . We comp t-tests . For eacWe compared biometrical measurements made on fifty study skins in the National Zoological Museum, Institute of Zoology, Chinese Academy of Sciences, including 13 specimens from Mongolia and Inner Mongolia, 18 from the eastern Himalayas and 19 from Central/East China. The lengths of the wing, tail, tarsus and bill were measured using a digital caliper. Body masses and body lengths were taken from the field collection records. Differences in the measurements between the three groups of the great tits were analyzed using a one-way analysis of variance .Accession codes: Sequencing data for the great tit have been deposited in Short Read Archive under project number PRJNA SRP274877.How to cite this article: Qu, Y. et al. Genetic responses to seasonal variation in altitudinal stress: whole-genome resequencing of great tit in eastern Himalayas. Sci. Rep.5, 14256; doi: 10.1038/srep14256 (2015).


The molecular mechanisms underlying chordoma pathogenesis are unknown. We therefore sought to identify novel mutations to better understand chordoma biology and to potentially identify therapeutic targets. Given the relatively high costs of whole genome sequencing, we performed a focused genetic analysis using matrix-assisted laser desorption/ionization-time of flight mass spectrometer (Sequenom iPLEX genotyping). We tested 865 hotspot mutations in 111 oncogenes and selected tumor suppressor genes (OncoMap v. 3.0) of 45 human chordoma tumor samples. Of the analyzed samples, seven were identified with at least one mutation. Six of these were from fresh frozen samples, and one was from a paraffin embedded sample. These observations were validated using an independent platform using homogeneous mass extend MALDI-TOF (Sequenom hME Genotyping). These genetic alterations include: ALK (A877S), CTNNB1 (T41A), NRAS (Q61R), PIK3CA (E545K), PTEN (R130), CDKN2A (R58*), and SMARCB1 (R40*). This study reports on the largest comprehensive mutational analysis of chordomas performed to date. To focus on mutations that have the greatest chance of clinical relevance, we tested only oncogenes and tumor suppressor genes that have been previously implicated in the tumorigenesis of more common malignancies. We identified rare genetic changes that may have functional significance to the underlying biology and potential therapeutics for chordomas. Mutations in CDKN2A and PTEN occurred in areas of chromosomal copy loss. When this data is paired with the studies showing 18 of 21 chordoma samples displaying copy loss at the locus for CDKN2A, 17 of 21 chordoma samples displaying copy loss at PTEN, and 3 of 4 chordoma samples displaying deletion at the SMARCB1 locus, we can infer that a loss of heterozygosity at these three loci may play a significant role in chordoma pathogenesis. Chordoma is an aggressive primary malignancy of the axial skeleton that is thought to originate from notochordal tissueThe identification of driver mutations in malignancy, such as EGFR-mutated lung cancers, c-Kit mutated gastrointestinal stromal tumors, and ALK-translocated tumors, among others, has dramatically changed the treatment landscape for these diseases Karyotype studies observed chordomas with loss at chromosome 1p and 3p and gains involving chromosomes 7q, 20, 5q, and 12q http://www.massgeneral.org/cancer/research/resourcelab.aspx?id=31).Institutional review board approval was obtained to retrospectively study these tumor samples from the Partners Human Research Committee, 116 Huntington Avenue, Suite 1002, Boston, MA 02116. The protocol number is 2007P-002464. The institutional review board waived the need to consent. Tumor specimens were obtained from the clinical archives of Dr. Francis Hornicek  and the Massachusetts General Hospital Tissue Repository .Tumor specimens were obtained from the clinical archives of Dr. Francis Hornicek  and the Massachusetts General Hospital Tissue Repository (Extraction of DNA from chordoma tumor tissues and cell lines were performed using QIAamp DNA Micro kit (Qiagen) as previously described in prior publications Selection of cancer gene mutations for assay design and mass spectrometric genotyping were performed as previously described Primers for PCR amplification and the extension probe were designed using Sequenom MassARRAY Assay Design 3.0 and the resulting DNA sequences were (1) queried in the dbSNP database to avoid incorporation of SNPs during assay design, (2) confirmed through a BLAST-like alignment tool (BLAT) and modified where necessary to avoid pseudogene amplificationPrimers and probes were pooled and then validated on control DNA derived from the CEPH panel of human HapMap DNAs (Coriell Institute) as well as a panel of human cell lines with known mutational status, as described previouslyCandidate mutations were further filtered by manual review and selected for confirmation using multi-base extension homogenous Mass-Extend (hME) chemistry with plexing of ≤6 assays per pool. Conditions for hME validation were consistent with the methods described by MacConaill et al. 2009 Array CGH using the Agilent 4x180k CGH + SNP microarray  was previously described A total of 45 DNA samples were derived from 40 patients who had undergone operative resections of their chordoma. Patient date of surgery (DOS), age, sex, tumor location, recurrence, and metastasis are catalogued in Examples of the mass spectrometric readouts are shown in Two samples (samples #7 and 8 in ), taken from the same patient at different anatomic locations and surgical dates, each had the same mutations in both SMARCB1 and CDKN2A. When we performed CGH analysis on the samples with CDKN2A and PTEN mutations, we observed copy number losses at respective loci  was identified. Similar to our previously reported rates of detecting similar frequency of mutations between freshly frozen and paraffin embedded osteosarcoma tumor samples ive loci , demonstChordomas have been previously described to have chromosomal aberrations and are characterized by chromosomal gains and losses at various regions throughout the genome With current technology, whole-genome sequencing of large collections of chordomas is costly and laborious. Thus, we aimed to broaden our mutational screen while focusing only on those genetic alterations that have been previously implicated as tumorigenisis drivers in other tumor types with the overall goal of identifying new mutations that could direct further research into chordoma therapeutic discovery. Some of the mutations identified in this study are in genes well-known for their role as tumor suppressors, such as PTEN and CDKN2A. Interestingly, both of these genes lie in chromosomal regions that were frequently found to have copy number losses in the recent CGH array analysis (9p21 for CDKN2A and 10q23.3 for PTEN) at a rate of >80% for each. Therefore, we performed CGH in the samples containing mutations in PTEN and CDKN2A and found chromosomal loss at each loci in the respective samples. Interestingly, 33% of samples tested in Le et al. collection found complete loss of both copies of CDKN2A – arguing that either a point mutation at CDKN2A or complete copy loss of CDKN2A can lead to LOH at this locus SMARCB1 is a subunit of the ATP-dependent chromatin remodeling complex SWI/SNF, a powerful epigenetic tumor suppressor, which directly antagonizes the histone methyltransferase EZH2. Mutations in SWI/SNF members are increasingly being recognized in malignancies where they are now thought by some groups to be more common than inactivating mutations in p53 These tumors, like chordomas, are groups of rare neoplasms for which no effective therapy is known. Mobley et al. observed that in poorly differentiated chordomas, expression of SMARCB1 is also lacking. They used FISH to evaluate the SMARCB1 locus at chromosome 22q and found deletion in 3 of 4 samplesSeveral of the mutations found in our study, such as ALK, PIK3CA, and CTNNB1, each have inhibitors that are either commercially available, such as the ALK inhibitor XALKORI (crizotinib), or have multiple inhibitors currently under active clinical developmentIn conclusion, we observed chordomas with point mutations in tumor suppressor genes in areas of frequent chromosomal loss and in oncogenes with commercially available inhibitors. The former suggests possible mechanisms for chordoma tumorigenesis, the latter suggests possibilities for novel therapies. Further studies need to be done in order to establish these genes as important biomarkers or targets in chordoma. The ideal study would be a complete genome sequencing effort involving many more chordoma samples. We hope that this initial sign of successful genotyping in the largest collection of human chordoma tumor samples to date may ignite enthusiasm to further investigations into the molecular pathology of this disease that currently has no effective medical therapy.


Furthermore, expression of the neural markers NOS, VIP and GFAP also increased during culture of aganglionic gut neurospheres which we show can be transplantation into cultured embryonic mouse gut explants to restore a normal frequency of contractility. To determine the origin of the progenitor cells in aganglionic region, we used fluorescence-activated cell sorting to demonstrate that only p75-positive neural crest-derived cells present in the thickened nerve trunks characteristic of the aganglionic region of Hirschsprung gut gave rise to neurons in culture. The derivation of enteric nervous system progenitors in the aganglionic gut region of Hirschprung’s patients not only means that this tissue is a potential source of cells for future autologous transplantation, but it also raises the possibility of inducing the differentiation of these endogenous cells in situ to compensate for the aganglionosis.Enteric nervous system progenitor cells isolated from postnatal human gut and cultured as neurospheres can then be transplanted into aganglionic gut to restore normal patterns of contractility. These progenitor cells may be of future use to treat patients with Hirschprung’s disease, a congenital condition characterized by hindgut dysmotility due to the lack of enteric nervous system ganglia. Here we demonstrate that progenitor cells can also be isolated from aganglionic gut removed during corrective surgery for Hirschsprung’s disease. Although the enteric nervous system marker calretinin is not expressed in the aganglionic gut region,  During embryonic development, the enteric nervous system (ENS) is mainly derived from cells originating in the vagal region of the neural crest which migrate caudally to colonize the whole length of the gut . During ENSPC have been isolated from the gut of both embryonic and postnatal rodent embryos, and also from postnatal human bowel . When cuin situ [The objectives of work on ENSPC are both to elucidate fundamental mechanisms of development and to facilitate translational research aimed at the development of clinical applications. Specifically, ENSPC present novel opportunities to treat Hirschsprung’s disease , which iin situ . Consequin situ . To thisin situ ,15.Before any such transplantation therapy is attempted, several crucial questions with regard to efficacy and safety need to be answered. For example, ENSPC proliferate readily in culture but we do not know if they will revert to a quiescent state after transplantation, or alternatively if will be necessary use differentiated cells derived from them for transplantation . Furtherdemonstrate present evidence that ENSPC from the aganglionic gut arise from cells of the thickened extrinsic nerve trunks characteristic of the aganglionic gut regions in Hirschsprung’s disease. These observations have implications both for our understanding of ENS developmental mechanisms involved in the pathogenesis of Hirschsprung’s disease, and also for the development of novel therapies. Thus, cells from aganglionic Hirschsprung gut might be used for autologous transplantation, or alternatively neurogenesis might be stimulated within the aganglionic segment in situ, thereby removing the need for surgery.To address these questions, we have begun to undertake an in depth study of the origins and behavior of ENSPC obtained from Hirschsprung patients. Surprisingly, we found that ENSPC-like cells can be isolated from the aganglionic region of Hirschsprung gut. Additionally, we Ethical approval for the isolation of human ENS progenitor cells was given by the UK North West 3 Research Ethics Committee (Ref: 10/H1002/77). Written parental consent was obtained before samples were taken. Both ganglionic and aganglionic human gut specimens were obtained from 12 patients (age range 1–6 months) with short-segment Hirschsprung’s disease, and 1 patient with the long segment form of the disease. Frozen tissue sections were examined at the time of surgery by a clinical pediatric pathologist to establish the absence of enteric ganglia and the presence of thickened nerve trunks characteristic of affected bowel.The use of mouse tissue in this study was approved by the Animal Welfare Committee of the University of Liverpool which is the Institutional Animal Care and Use Committee (IACUC). The University of Liverpool is a Licenced Establishment in accordance with the United Kingdom Animal (Scientific Procedures) Act of 1986. Pregnant mice were sacrificed by exposure to carbon dioxide gas in a rising concentration, followed by dislocation of the neck, followed by decapitation of 11.5 days post-coitum embryos, which are appropriate methods under Schedule 1 of the Act. The principle of refinement (Section 2A of the Act) requires that mice were humanely killed with a minimum of pain, suffering and distress, by a person with appropriate training, registered as competent to kill animals for this project according to Schedule 1 of the Act in the register kept by the Animal Welfare Committee (IACUC) of the University of Liverpool. The study did not require a Home Office project license because no in vivo experiments or regulated procedures as defined by the Act were carried out.2 full-thickness ganglionic and aganglionic human gut specimens were obtained from 12 patients (age range 1–6 months) with short-segment Hirschsprung’s disease, and 1 patient with the long segment form of the disease. Tissue for cell culture was transferred to the laboratory on ice, wrapped in sterile, saline-soaked gauze. Single cell suspensions were obtained from human ganglionic and aganglionic colonic biopsies as previously described [3 pieces and placed in 1% (w/v) Dispase and 1% (w/v) Collagenase IV  for a total of 2 h. The samples were triturated every 15 min using a 5ml pipette and the single cell suspensions were removed from remaining clumps after each trituration. The single cell suspensions were combined and centrifuged before resuspension in culture media. Typical yields from each specimen were 3–6 x 106 cells.One cmescribed ,15. Brie5 and 106 cells dissociated from Hirschsprung ganglionic gut were plated into 6cm Nunc tissue culture dishes and incubated at 37°C in a humidified 5% CO2 incubator. Media were refreshed every 3 days by careful aspiration of 50% of the volume in each dish and replacement with the same volume of fresh media. Under these conditions neurospheres typically formed between 7–14 days. The loosely attached neurospheres were harvested every 14–15 days by detachment from the more strongly attached cells on the culture substratum by pipetting a stream of medium over them. The cells were then passaged be digesting the neurospheres with 0.05% (w/v) trypsin and mechanical dissociation by trituration to produce single cell suspensions for further couture to form secondary and tertiary neurospheres [To ensure optimal neurosphere culture conditions, we compared the culture medium previously used by our group and others (Dulbecco’s modified Eagle medium low (1% w/v) glucose, supplemented with 1% (v/v) fetal calf serum, 20 ng/mL FGF2 and 20 ng/mL EGF) with a medium lacking added growth factors consisting of Dulbecco’s modified Eagle medium , supplemented with 199 media (20% v/v), heat-inactivated horse serum (7% v/v), 100units/ml penicillin and 100 μg/ml streptomycin . Between 5x10ospheres . The neu® (Dako) was then applied for 30 min. Negative controls omitted the primary antibody and positive controls were histologically normal rectal biopsies. Paraffin-embedded sections from approximately half the cohort of patients were examined for calretinin staining, and in all cases this staining confirmed the original pathological diagnosis of Hirschsprungs aganglionic bowel made at surgery. Furthermore, there were no discrepancies between the clinical diagnosis of aganglionosis and the confirmatory observations described in this paper.Clinical diagnosis of all ganglionic and aganglionic bowel segments was established at surgery and routine H&E staining of frozen biopsy sections. The diagnosis was also confirmed after surgery by immunohistochemistry for calretinin on paraffin wax-embedded, 4 μm-thick sections of rectal biopsies , using a® secondary antibodies (Life Technologies) were all used at 1:1000.For immunofluorescence, samples for frozen tissue sections were fixed at time of surgery in 4% (w/v) paraformaldehyde and embedded in Shandon Cryomatix (Thermo Fischer Scientific). Seven μm serial frozen sections were prepared from full thickness colonic biopsies. Cells dissociated from fresh tissue or neurospheres were either spun down onto slides using a Shandon Cytospin3  or fixed in situ with 4% (w/v) paraformaldehyde after culture. Primary antibodies used for immunofluorescence were used at the following concentrations: rabbit anti-calretinin (Abcam ab702) 1:25; mouse anti- GFAP (Sigma-Aldrich G3893) 1:250; mouse anti-p75 (Abcam ab3125) 1:1000; rabbit anti-neuronal nitric oxide synthase  1:200; mouse anti-smooth muscle actin  1:200; rabbit anti-S100 (Abcam ab868) 1:500; mouse anti-β tubulin III  1:1000; mouse anti-human specific ribonucleoprotein  1:50. Alexafluor6 cells were then incubated on ice for 30 min either with 1:1000 mouse anti-p75 antibody (Abcam ab3125) or isotype control antibodies in 200μl FACS buffer. Cells were then centrifuged and re-suspended in 200μl FACS buffer with 1:1000 Alexafluor 488® goat anti-mouse secondary antibodies. After 45 minutes the cells were re-suspended in 1ml FACS buffer and sorted using FACSaria® (BD Biosciences). Cell aliquots from p75-positive and-negative subpopulations were then cultured in poly-D-lysine/laminin-coated 8 well chamber slides (Thermo Scientific) for 6 days, before assessing neuronal differentiation by Tuj1 immunofluorescence.Single cell suspensions from fresh bowel samples were transferred to ice-cold FACS buffer . 5 x 10ex vivo assessment of gut contractility together with observation of cell migration and phenotype was as previously described using aganglionic distal colon explants dissected from 11.5 days post coitum CD-1 mouse embryos [Timed pregnant CD-1 mice were purchased from Charles River (UK) Ltd., . The  embryos . Positiv embryos . Explant embryos . After m® 4.03 was used to analyse data. Non-parametric continuous data were analysed using Mann-U Whitney, a two-tailed t test was used for parametric data 1-way ANOVA analysis with Tukeys correction was used for parametric grouped data and a Krustal-Wallis test for non parametric data. Standard error of the mean was used for comparing outcomes of repeated experiments. P < 0.05 was taken as significant.GraphPad PrismThe ganglionic and aganglionic regions of Hirschprung colon were confirmed by immunohistology of biopsies using the ENS marker calretinin which is absent from the aganglionic gut see . ConsistCells isolated from all 12 short segment aganglionic Hirschsprung samples used in this study behaved similarly when brought into culture: during the first days of culture cells began to clump together to form loosely attached neurosphere-like bodies , similarIn order to characterize neural cell development in the cultures from aganglionic Hirschsprung gut tissue, we compared quantitatively the development of specific phenotypes from ganglionic and aganglionic gut cells immediately after initial dissociation and during culture as neurospheres. As expected, the majority of cells isolated directly from both ganglionic and aganglionic gut initially expressed smooth muscle actin, however, within the primary neurospheres harvested after the first 10 days of neurosphere culture their proportion dropped markedly during the first 10 days of neurosphere culture, and subsequently remained at low levels . In contThe specific ENS marker calretinin was initially only expressed by very low numbers of cells isolated from ganglionic gut, and as expected see  it was uGiven the presence of p75 positive cells in the thickened nerve trunks of aganglionic Hirschsprung gut, we hypothesized that these are the precursors of the cells expressing neural markers found in increasing proportions in the neurosphere cultures . To demoex vivo aganglionic embryonic mouse gut explant model we have previously used to test cells from ganglionic gut [To test the functional potential of neural progenitors derived from aganglionic Hirschprung gut, neurospheres were transplanted into the onic gut . The agaThe pattern of gut contractility was determined using a previously described optical motion capture technique . ContracIn this study we show that ENSPC-like cells can be isolated from the aganglionic gut of patients with Hirschsprung disease. The cells of neurospheres derived from the aganglionic gut are demonstrated to have the same phenotypes and functional properties as those previously described for ENSPC isolated from the ganglionic gut. Thus, they can differentiate into neuronal and glial cells ,6,10, miAganglionic Hirschsprung bowel is characterized histopathologically not only by the absence of calretinin-positive ENS ganglia , but alsThe migratory properties of sacral neural crest cells differ from those of vagal cells ,24, and in vivo using pulse-chase DNA labeling have provided evidence for the existence of a small population of extraganglionic cells able to colonize ganglia [It is now well established that the postnatal gut contains multipotent cells that are capable of differentiation into neurons and glia characteristic of the ENS ,11,12,26 ganglia . Our obsSchwann and other glial cells have been well-documented to show a high degree of plasticity during development. Thus, it has recently been shown that neurons of parasympathetic ganglia develop from Schwann cell progenitors migrating from preganglionic nerves during development ,29. Furtin vivo, removing the need for surgery.The fact that neuronal progenitors can be isolated from aganglionic Hirschsprung gut raises the possibility of using aganglionic tissue, normally discarded at surgery, to provide a source of cells for future autologous transplants in the treatment of Hirschsprung’s disease. Additionally, the existence of these cells raises the intriguing possibility to design future therapeutic modalities to stimulate neurogenesis in the aganglionic region S1 FigA) Phase contrast photomicrograph of neurosphere-like cell aggregate derived from Hirschsprung gut after culture for 15 days in medium containing horse serum (see (TIF)Click here for additional data file.S2 FigThe percentage of cells expressing p75 are shown from neurospheres cultured with either growth factor medium (GFM) or horse serum medium (HSM), and in the initial cell dissociate at time 0. Aliquots of cultured neurospheres were harvested at the times shown and single cell suspensions prepared by trypsinization and trituration. The cells were then allowed to attach to tissue culture slides before paraformaldehyde fixation and processing for p75 immunofluorescence. Immunofluorescent cells were counted using a 40x objective by systematically surveying rows across the surface of the slide, corresponding to 25% of the culture surface area. Numbers of p75-positive cells are expressed as a percentage of the total number of cells counted, which had been counterstained with DAPI. There is a continuous increase in the number of p75 positive cells with time in culture but there is no difference (P>0.45) in numbers of positive cells between the two media at any single time point (ANOVA). Error bars show SEM, n = 4.(TIF)Click here for additional data file.S3 FigThe presence and absence of ENS ganglia in full thickness paraffin embedded sections of colonic biopsies of (A) ganglionic, and (B) aganglionic bowel was confirmed by immunohistology for calretinin after surgery. Sections are counterstained with hematoxylin/eosin. Scale bars = 100μm.(TIF)Click here for additional data file.


Here, we report a thorough theoretical and experimental characterization of myosin-derived SHG intensity profiles within intact zebrafish skeletal muscle. We determined that “SHG vernier” patterns, regions of bifurcated SHG intensity, are illusory when sarcomeres are staggered with respect to one another. These optical artifacts arise due to the phase coherence of SHG signal generation and the Guoy phase shift of the laser at the focus. In contrast, two-photon excited fluorescence images obtained from fluorescently labeled sarcomeric components do not contain such illusory structures, regardless of the orientation of adjacent myofibers. Based on our results, we assert that complex optical artifacts such as SHG verniers should be taken into account when applying functional SHG imaging as a diagnostic readout for pathological muscle conditions.SHG microscopy is an emerging microscopic technique for medically relevant imaging because certain endogenous proteins, such as muscle myosin lattices within muscle cells, are sufficiently spatially ordered to generate detectable SHG without the use of any fluorescent dye. Given that SHG signal is sensitive to the structural state of muscle sarcomeres, SHG functional imaging can give insight into the integrity of muscle cells  SHG-derived contrast has a particularly attractive property relevant to biological imaging. The signal is tunable, meaning that specific illumination wavelengths can be chosen to minimize (i) background photons  as well as (ii) spectral overlap with other contrast agents being imaged in parallel 345Second harmonic generation (SHG) is a nonlinear optical process where two photons interact with a highly ordered, non-centrosymmetric material  to generate a single photon with half the wavelength or double the frequency of the incident photons1To produce detectable amounts of SHG signal, the SHG photons generated by different molecules must be in phase, leading to constructive interference of the signal. Certain components such as collagen, microtubules, and muscle myosin arrays possess sufficient order to fulfill these strict symmetry constraints. These biological structures generate detectable SHG signal, enabling them to be imaged in a label-free manner7The coherent nature of SHG has proven especially relevant when visualizing myosin bundles within the sarcomeres of striated muscle11Rana temporaria frog muscle fibers in the late 1950s121516One prominent SHG signal motif that is common in endogenous SHG imaging of skeletal muscle in both the zebrafish and the mouse are so-called “SHG verniers” — ‘Y’-shaped patterns and curved distortions in SHG signal between sarcomeric units in a myofiber . These vin silico. In this model, we manipulated the geometry of adjacent synthetic muscle cells to determine the possible SHG signal patterns that may arise when illuminated with focused laser light (see Methods). To act as a standard for comparison of SHG signal from the muscle myosin, we also added a fluorescent component to the model, specifically considering the myosin thick filaments to be fluorescently labeled in addition to being SHG capable. The simplified hypothetical muscle tissue in our model is made up of two adjacent myofibers with multiple sarcomeric units . Additionally, we assumed an NA of 0.8 for the objective and 0.55 for the condenser in our simulations, as these are reasonably standard values when performing high resolution laser scanning imaging in many biological applications.To investigate the source of SHG vernier patterns in muscle tissue, we constructed a simplified numerical simulation of an SHG microscopy experiment. We first applied our theoretical characterizations by modeling hypothetical muscle fibers ic units  organizeOur calculations yielded striking differences in TPEF  and SHG If verniers were in fact optical artifacts of the SHG process, we should be able to see differing signal in staggered muscle fibers when comparing SHG and TPEF images. Fluorescent emission from fluorescently labeled sarcomere components would show no increased fluorescence between fibers and would not have curvature or bifurcations in the signal, while SHG curvature and verniers would be prominent in the same imaging plane , dependiSince the model predicts that illusory SHG verniers have the potential to cross the plasma membrane boundaries of adjacent muscle cells . In this case, the myosin-derived SHG co-localizes with the TPEF from the fluorophore-conjugated secondary antibody . In long10Gt(tpm3-citrine)ct5a established by Trinh et al.In a second experimental condition, we wanted to ensure that the SHG verniers that we had seen were not an artifact of the myosin antibody labeling. In an effort to visualize labeled thin filaments within sarcomeres, we took advantage of the FlipTrap transgenic line To account for the complex endogenous SHG signal patterns that arise in whole zebrafish tissue, we expanded our initial simplified SHG model into arbitrary geometries. We aimed to recapitulate vernier patterns in real tissue, validating the expected differences in TPEF versus SHG signal seen in the original theoretical analysis . Our samWithin this volume, vernier pattern signal in the endogenous SHG channel  crosses In an effort to uncover the source of SHG verniers within intact muscles, we implemented a theoretical model, paying special attention to the consequences of the phase coherence of SHG propagation and the Guoy phase shift of a laser at the sample focus. Unlike fluorescent signal within the sarcomere, which always retains a banded pattern that is evenly spaced and unconnected, SHG signal patterns can be distorted, depending on the orientation of the incoming laser as well as the organization of adjacent muscle fibers. Our analysis of phase coherence and cancellation proved to be invaluable for discovering this optical artifact. Comparable phase considerations have benefited other nonlinear microscopic approaches, such as coherent anti-Stokes Raman scattering microscopy27Although our model predicts the occurrence of SHG verniers when adjacent sarcomeres are staggered, we do not rule out the possibility that some fraction of vernier patterns are a result of physical remodeling processes in the muscle cells, as has been previously shown in isolated rat and Xenopus muscle tissueZebrafish were raised, hatched, injected, and maintained in a house colony as previously describedGt(tpm3-citrine)ct5a (Danio rerio) embryos were obtained by incrossing males and females from the same genetic background. These embryos were allowed to develop for ~18–20 hours before being treated with 1 × 1-phenyl 2-thiourea  to inhibit pigment formation for the following few days of development. PTU medium was exchanged once every day or two. In the days before imaging, the embryos were kept in egg waterWild type (WT) or ine)ct5a  transgen2+/Mg2+-free 1× PBS 3 times . Gt(tpm3-citrine)ct5a zebrafish and the WT zebrafish were immediately prepared for imaging.To fix the zebrafish, the embryos/larvae were first anesthetized in 0.015–0.03% MS-222  and then placed in 4% methanol-free formaldehyde  on ice. After ∼5 min, embryos were allowed to continue to fix at room temperature on a nutator  for 1 hour before being washed in Ca2+/Mg2+-free) + 1% dimethyl sulfoxide (DMSO) + 1% bovine serum albumin (BSA) + 1.5% Triton X-100 detergent (referred to as PBSTB). Anti-sarcomere myosin  and anti-myomesin (mMaC Myomesin B4) primary antibodies were obtained from the Developmental Studies Hybridoma Bank at the University of Iowa. Primary antibodies were incubated on a nutator at room temperature in PBSTB with the whole-mount zebrafish for 2.25 hours (1:3 dilution). Embryos were washed 3 times (15 min each) in PBSTB before being suspended in PBSTB and treated with goat anti-mouse IgG secondary antibodies (1:200 dilution) conjugated to Alexa Fluor 488 nm . The embryos were incubated with the secondary antibody protected from light at room temperature on a nutator for 2.5 hours. After 3 washes (10 min each), the embryos were prepared for imaging.Embryos that were stained with antibody were first blocked overnight at 4 °C in 1× PBS  optical sections as images that are taken parallel to the long axis of the zebrafish, while “transverse” (x-z) optical sections are images that are taken perpendicular to the long axis of the zebrafish.Images were processed in Imaris , ImageJ (NIH), and Adobe Photoshop CS3 (Adobe Systems), where minor linear brightness/contrast adjustments and filtering  were performed.From the large 3D volume multichannel  datasets, sub-volumes were extracted that contained both vernier-pattern SHG signal and appreciable TPEF signal. All of the subsequent algorithms for segmentation and estimation of the surface normal vectors were performed using a script written in MATLAB . Implementation of the full 3D theoretical model to recapitulate the experimental SHG data using the sarcomeric TPEF signal was performed using a custom script in the open source Julia language . Poisson distributed background noise was simulated in the model data using a custom MATLAB script.Because SHG verniers are often found deep within the muscle compartments within zebrafish somites where light and antibody penetration is reduced, TPEF signal emanating from the immunostained sarcomeric components was low. The resulting low signal-to-noise ratio TPEF images were difficult to process using automatic segmentation algorithms, so we instead took advantage of a segmentation toolkit to perform manual segmentation of the dataHow to cite this article: Dempsey, W. P. et al. Determination of the source of SHG verniers in zebrafish skeletal muscle. Sci. Rep.5, 18119; doi: 10.1038/srep18119 (2015).


TERT gene has recently been identified as a susceptibility factor for myeloproliferative neoplasms (MPN) in the Icelandic population. Here, we evaluate the role of TERT rs2736100_C in sporadic and familial MPN in the context of the previously identified JAK2 GGCC predisposition haplotype. We have confirmed the TERT rs2736100_C association in a large cohort of Italian sporadic MPN patients. The risk conferred by TERT rs2736100_C is present in all molecular and diagnostic MPN subtypes. TERT rs2736100_C and JAK2 GGCC are independently predisposing to MPN and have an additive effect on disease risk, together explaining a large fraction of the population attributable fraction (PAF = 73.06%). We found TERT rs2736100_C significantly enriched (P = 0.0090) in familial MPN compared to sporadic MPN, suggesting that low-penetrance variants may be responsible for a substantial part of familial clustering in MPN. Am. J. Hematol. 89:1107–1110, 2014. © 2014 The Authors. American Journal of Hematology published by Wiley Periodicals, Inc.The C allele of the rs2736100 single nucleotide polymorphism located in the second intron of the  JAK2, most notably JAK2-V617F are present in all three disease subtypes, more frequently in PV MPL and CALR mutations are exclusively found in ET and PMF patients JAK2 locus has been found to predispose to JAK2 mutation positive sporadic and familial MPN TERT gene as risk variant for MPN in the Icelandic population TERT association in an independent cohort from a different ethnic background and evaluate the role of the TERT predisposition locus in familial clustering of MPN. Furthermore, we were testing the possibility of an interaction of TERT and JAK2 susceptibility loci in sporadic and familial MPN.Myeloproliferative neoplasms (MPN) constitute a group of phenotypically diverse chronic myeloid malignancies including three major disease entities: polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Mutually exclusive oncogenic somatic mutations in three genes have been identified in more than 90% of MPN cases n= 717), familial MPN (n= 121) and control (n= 202) subjects from Italy were obtained after written informed consent. The study was approved by the institutional ethics committee  and procedures were in accordance with the Helsinki declaration. Details on patient characteristics and sample collection have been described in a previous study Blood samples from sporadic MPN  and rs10974944 (JAK2) was performed using commercially available TaqMan SNP genotyping assays .Presence of the TERT and JAK2 loci were calculated as previously described by others TERT/JAK2 genotypic classes was calculated using logistic regression.Statistical analyses were performed using the R statistical software (version 3.0.3) n= 717) of a different ethnic background , however, statistical significance is absent, possibly due to low sample sizes (Supporting Information Table 1). As in the prior study TERT rs2736100_C association was present in PV, ET, and PMF, implying a general role in MPN pathogenesis (Supporting Information Table 1).We confirmed the previously reported TERT and JAK2 as well as their possible interaction. Conditional logistic regression analysis revealed that TERT and JAK2 loci are independently predisposing to MPN, the total combined risk approximating the sum of the two genotypes . However, MPN families consisting of both JAK2-positive and JAK2-negative members might be confounding the possible association, as JAK2 GGCC predisposes mainly to JAK2-positive MPN. Indeed, when restricting analysis to families exclusively consisting of JAK2-positive members, we observed a significant trend toward JAK2 GGCC enrichment in familial MPN compared to sporadic MPN . Moreover, the familial MPN cohort exhibits enrichment for individuals carrying more than three risk alleles  and a trend for odds ratios increasing toward double positive (TERT/JAK2) individuals (Supporting Information Table 2), further supporting the possibility of JAK2 GGCC contributing to MPN familial clustering. Independence of TERT and JAK2 loci in mediating MPN susceptibility, as observed in sporadic MPN, is also true for familial MPN , they explain a large part (combined PAF = 73.06%) of the population susceptibility for MPN. The absolute risk for developing MPN in different TERT/JAK2 genotypic classes is calculated in Supporting Information Table 5. As the risk variant rs2736100_C is present at high frequency  in the general population.Similar to that reported in the Icelandic study TERT gene encodes the reverse transcriptase of the telomerase complex, essential for maintaining telomere length TERT expression. TERT rs2736100_C was previously shown to also associate with elevated risk for several other cancers, albeit with lower effect The TERT rs2736100_C for all MPN subtypes as well as JAK2 GGCC for JAK2-positive MPN exhibit effect sizes stronger than typically observed for common cancer-predisposing variants TERT rs2736100_C (TableJAK2 GGCC in familial clustering remains to be confirmed in a larger set of JAK2-positive MPN families.Enrichment of common susceptibility loci in familial forms of predominantly sporadic cancers has been reported previously TERT and JAK2 loci explains most of the population risk for developing MPN. Enrichment of the TERT risk variant in familial MPN suggests the possibility of random accumulation of several common high frequency variants being responsible for parts of the elevated risk underlying familial clustering. More common low penetrance and/or higher penetrance rare mutations remain to be discovered to explain the missing heritability in sporadic MPN and the missing excess familial risk in MPN.In conclusion, common variation at