Hematopoietic Stem Cell Development and Transcriptional Regulation

The continuous production of blood cells, a process termed hematopoiesis, is sustained throughout the lifetime of an individual by a relatively small population of cells known as hematopoietic stem cells (HSCs). HSCs are unique cells characterized by their ability to self-renew and give rise to all types of mature blood cells. Given their high proliferative potential, HSCs need to be tightly regulated on the cellular and molecular levels or could otherwise turn malignant. On the other hand, the tight regulatory control of HSC function also translates into difficulties in culturing and expanding HSCs in vitro. In fact, it is currently not possible to maintain or expand HSCs ex vivo without rapid loss of self-renewal. Increased knowledge of the unique features of important HSC niches and of key transcriptional regulatory programs that govern HSC behavior is thus needed. Additional insight in the mechanisms of stem cell formation could enable us to recapitulate the processes of HSC formation and self-renewal/expansion ex vivo with the ultimate goal of creating an unlimited supply of HSCs from e.g. human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPS) to be used in therapy. We thus asked: How are hematopoietic stem cells formed and in what cellular niches does this happen (Papers I, II)? What are the molecular mechanisms that govern hematopoietic stem cell development and differentiation (Papers III, IV)? Importantly, we could show that placenta is a major fetal hematopoietic niche that harbors a large number of HSCs during midgestation (Paper I)(Gekas et al., 2005). In order to address whether the HSCs found in placenta were formed there we utilized the Runx1-LacZ knock-in and Ncx1 knockout mouse models (Paper II). Importantly, we could show that HSCs emerge de novo in the placental vasculature in the absence of circulation (Rhodes et al., 2008). Furthermore, we could identify defined microenvironmental niches within the placenta with distinct roles in hematopoiesis: the large vessels of the chorioallantoic mesenchyme serve as sites of HSC generation whereas the placental labyrinth is a niche supporting HSC expansion (Rhodes et al., 2008). Overall, these studies illustrate the importance of distinct milieus in the emergence and subsequent maturation of HSCs. To ensure proper function of HSCs several regulatory mechanisms are in place. The microenvironment in which HSCs reside provides soluble factors and cell-cell interactions. In the cell-nucleus, these cell-extrinsic cues are interpreted in the context of cell-intrinsic developmental programs which are governed by transcription factors. An essential transcription factor for initiation of hematopoiesis is Scl/Tal1 (stem cell leukemia gene/T-cell acute leukemia gene 1). Loss of Scl results in early embryonic death and total lack of all blood cells, yet deactivation of Scl in the adult does not affect HSC function (Mikkola et al., 2003b. In order to define the temporal window of Scl requirement during fetal hematopoietic development, we deactivated Scl in all hematopoietic lineages shortly after hematopoietic specification in the embryo . Interestingly, maturation, expansion and function of fetal HSCs was unaffected, and, as in the adult, red blood cell and platelet differentiation was impaired (Paper III)(Schlaeger et al., 2005). These findings highlight that, once specified, the hematopoietic fate is stable even in the absence of Scl and is maintained through mechanisms that are distinct from those required for the initial fate choice. As the critical downstream targets of Scl remain unknown, we sought to identify and characterize target genes of Scl (Paper IV). We could identify transcription factor Mef2C (myocyte enhancer factor 2 C) as a novel direct target gene of Scl specifically in the megakaryocyte lineage which largely explains the megakaryocyte defect observed in Scl deficient mice. In addition, we observed an Scl-independent requirement of Mef2C in the B-cell compartment, as loss of Mef2C leads to accelerated B-cell aging (Gekas et al. Submitted). Taken together, these studies identify key extracellular microenvironments and intracellular transcriptional regulators that dictate different stages of HSC development, from emergence to lineage choice to aging.

Introduction of SV40ER and hTERT into mammospheres generates breast cancer cells with stem cell properties

Emerging evidence suggests that cancers arise in stem/progenitor cells. Yet, the requirements for transformation of these primitive cells remains poorly understood. In this study, we have exploited the `mammosphere' system that selects for primitive mammary stem/progenitor cells to explore their potential and requirements for transformation. Introduction of Simian Virus 40 Early Region and hTERT into mammosphere-derived cells led to the generation of NBLE, an immortalized mammary epithelial cell line. The NBLEs largely comprised of bi-potent progenitors with long-term self-renewal and multi-lineage differentiation potential. Clonal and karyotype analyses revealed the existence of heterogeneous population within NBLEs with varied proliferation, differentiation and sphere-forming potential. Significantly, injection of NBLEs into immunocompromised mice resulted in the generation of invasive ductal adenocarcinomas. Further, these cells harbored a sub-population of CD44(+)/CD24(-) fraction that alone had sphere- and tumor-initiating potential and resembled the breast cancer stem cell gene signature. Interestingly, prolonged in vitro culturing led to their further enrichment. The NBLE cells also showed increased expression of stemness and epithelial to mesenchymal transition markers, deregulated self-renewal pathways, activated DNA-damage response and cancer-associated chromosomal aberrations-all of which are likely to have contributed to their tumorigenic transformation. Thus, unlike previous in vitro transformation studies that used adherent, more differentiated human mammary epithelial cells our study demonstrates that the mammosphere-derived, less-differentiated cells undergo tumorigenic conversion with only two genetic elements, without requiring oncogenic Ras. Moreover, the striking phenotypic and molecular resemblance of the NBLE-generated tumors with naturally arising breast adenocarcinomas supports the notion of a primitive breast cell as the origin for this subtype of breast cancer. Finally, the NBLEs represent a heterogeneous population of cells with striking plasticity, capable of differentiation, self-renewal and tumorigenicity, thus offering a unique model system to study the molecular mechanisms involved with these processes. Oncogene (2012) 31, 1896-1909; doi:10.1038/onc.2011.378; published online 29 August 2011

Brf1 post-transcriptionally regulates pluripotency and differentiation responses downstream of Erk MAP Kinase

FGF/Erk MAP Kinase Signaling is a central regulator of mouse embryonic stem cell (mESC) self-renewal, pluripotency and differentiation. However, the mechanistic connection between this signaling pathway activity and the gene circuits stabilizing mESCs in vitro remain unclear. Here we show that FGF signaling post-transcriptionally regulates the mESC transcription factor network by controlling the expression of Brf1 (zfp36l1), an AU-rich element mRNA binding protein. Changes in Brf1 level disrupts the expression of core pluripotency-associated genes and attenuates mESC self-renewal without inducing differentiation. These regulatory effects are mediated by rapid and direct destabilization of Brf1 targets, such as Nanog mRNA. Interestingly, enhancing Brf1 expression does not compromise mESC pluripotency, but does preferentially regulate differentiation to mesendoderm by accelerating the expression of primitive streak markers. Together, these studies demonstrate that FGF signals utilize targeted mRNA degradation by Brf1 to enable rapid post-transcriptional control of gene expression.

MicroRNA-132 is a physiological regulator of hematopoietic stem cell function and B-cell development

MicroRNAs are a class of small non-coding RNAs that negatively regulate gene expression. Several microRNAs have been implicated in altering hematopoietic cell fate decisions. Importantly, deregulation of many microRNAs can lead to deleterious consequences in the hematopoietic system, including the onset of cancer, autoimmunity, or a failure to respond effectively to infection. As such, microRNAs fine-tune the balance between normal hematopoietic output and pathologic consequences. In this work, we explore the role of two microRNAs, miR-132 and miR-125b, in regulating hematopoietic stem cell (HSC) function and B cell development. In particular, we uncover the role of miR-132 in maintaining the appropriate balance between self-renewal, differentiation, and survival in aging HSCs by buffering the expression of a critical transcription factor, FOXO3. By maintain this balance, miR-132 may play a critical role in preventing aging-associated hematopoietic conditions such as autoimmune disease and cancer. We also find that miR-132 plays a critical role in B cell development by targeting a key transcription factor, Sox4, that is responsible for the differentiation of pro-B cells into pre-B cells. We find that miR-132 regulates B cell apoptosis, and by delivering miR-132 to mice that are predisposed to developing B cell cancers, we can inhibit the formation of these cancers and improve the survival of these mice. In addition to miR-132, we uncovered the role of another critical microRNA, miR-125b, that potentiates hematopoietic stem cell function. We found that enforced expression of miR-125b causes an aggressive myeloid leukemia by downregulation of its target Lin28a. Importantly, miR-125b also plays a critical role in inhibiting the formation of pro-B cells. Thus, we have discovered two microRNAs with important roles in regulating normal hematopoiesis, and whose dregulation can lead to deleterious consequences such as cancer in the aging hematopoietic system. Both miR-132 and miR-125b may therefore be targeted for therapeutics to inhibit age-related immune diseases associated with the loss of HSC function and cancer progression.

Lymphangioleiomyomatosis Biomarkers Linked to Lung Metastatic Potential and Cell Stemness

Lymphangioleiomyomatosis (LAM) is a rare lung-metastasizing neoplasm caused by the proliferation of smooth muscle-like cells that commonly carry loss-of-function mutations in either the tuberous sclerosis complex 1 or 2 (TSC1 or TSC2) genes. While allosteric inhibition of the mechanistic target of rapamycin (mTOR) has shown substantial clinical benefit, complementary therapies are required to improve response and/or to treat specific patients. However, there is a lack of LAM biomarkers that could potentially be used to monitor the disease and to develop other targeted therapies. We hypothesized that the mediators of cancer metastasis to lung, particularly in breast cancer, also play a relevant role in LAM. Analyses across independent breast cancer datasets revealed associations between low TSC1/2 expression, altered mTOR complex 1 (mTORC1) pathway signaling, and metastasis to lung. Subsequently, immunohistochemical analyses of 23 LAM lesions revealed positivity in all cases for the lung metastasis mediators fascin 1 (FSCN1) and inhibitor of DNA binding 1 (ID1). Moreover, assessment of breast cancer stem or luminal progenitor cell biomarkers showed positivity in most LAM tissue for the aldehyde dehydrogenase 1 (ALDH1), integrin-beta 3 (ITGB3/CD61), and/or the sex-determining region Y-box 9 (SOX9) proteins. The immunohistochemical analyses also provided evidence of heterogeneity between and within LAM cases. The analysis of Tsc2-deficient cells revealed relative over-expression of FSCN1 and ID1; however, Tsc2-deficient cells did not show higher sensitivity to ID1-based cancer inhibitors. Collectively, the results of this study reveal novel LAM biomarkers linked to breast cancer metastasis to lung and to cell stemness, which in turn might guide the assessment of additional or complementary therapeutic opportunities for LAM.

Comparative Analysis of Five Different Homologous Feeder Cell Lines in the Ability to Support Rhesus Embryonic Stem Cells

我们以前的研究建立了五株猕猴饲养层细胞系来支持猕猴胚胎干细胞(rESCs)的生长:一岁猴耳皮肤成纤维细胞(MESFs)、两岁猴输卵管成纤维细胞(MOFs)、成年猴卵泡颗粒成纤维样细胞(MFGs)、成年猴卵泡颗粒上皮样细胞(MFGEs),以及MESFs的克隆成纤维细胞(CMESFs).我们发现MESFs、CMESFs、MOFs和MFGs,而不足MFGEs支持猕猴胚胎干细胞(rESCs,rhesus embryonic stem cells)的生长.通过半定量PCR的方法,我们在支持性的饲养层细胞中检测到了一些基因的高表达.在本研究中,我们运用Affymetrix公司的GeneChip Rhesus Macaque Genome Array芯片来研究这五株同源饲养层的表达谱,希望发现哪些细胞因子和信号通路在维持rESCs中起到重要作用.结果表明,除MFGE外,包括GREM2、bFGF,、KITLG,、DKK3、GREM1、AREG、SERPINF1和LTBF1等八个基因的mRNA在支持性的饲养层细胞中高表达.本研究结果提示,很多信号通路在支持rESCs的未分化生长和多潜能性方面可能起到了冗余的作用.

饲养层FGF2表达量对猕猴胚胎干细胞自我更新及多能性的影响

用转基因和RNA干扰(RNAi)法建立5组不同成纤维细胞生长因子-2(fibroblast growth factor-2,FGF2)表达量的猕猴耳部皮肤成纤维细胞(MESF)系:过表达FGF2组(f1),过表达的阴性对照组(f2),FGF2 RNA干扰组(f3),RNA干扰的阴性对照组(f4)和未作任何处理的对照组(f5).5组MESF的FGF2表达量相对值为f1:f2:f3:f4:f5=4:2:1:2:2;c-fos,TGF-β1,INHBA,Gremlinl在f1中表达量上升,在f3中表达量下降;BMP4,TGF-β2在f1中表达量下降,在f3中表达量上升;表明内源FGF2能够作用于MESF的TGF-β信号通路,引起相关基因表达量的变化.用这砦细胞作为饲养层长期培养(10代)猕猴胚胎干细胞(RhESC),结果在f1上培养的RhESC增殖速度都比对照组快,并且c-fos,TGF-β1,INHBA,Gremlinl,Oct-4,Nanog,Sox2表达量均上升,BMP4表达下调;在f3上培养的RhESC增殖较慢,BMP4表达上调,c-fos,TGF-β1,INHBA,Gremlinl,Oct-4,Nanog,Sox2表达下调.5组MESF上培养的RhESC形成的EB均表达各胚层早期标记基因(marker),说明RhESC的多能性没有受到影响,但表达量有差异,f1上RhESC形成的EB所有marker都低表达.结果表明饲养层的FGF2含量不仅影响自身相关基凶的表达,还对RhESC的自我更新有一定的作用.

不同FGF-2表达量的饲养层对猕猴和兔胚胎干细胞的增殖和自我更新的影响

胚胎干细胞（ESC）培养是ESC研究的基础，饲养层的选择是ESC培养的一个重要方面。本实验曾对不同的猕猴饲养层进行研究，显示能够更好的支持猕猴ESC生长的饲养层FGF-2表达量也较高。FGF-2，又名bFGF（碱性成纤维生长因子），是ESC生长所需的重要因子，但其中的分子机制现在并未完全了解。本文一方面对ESC相关研究进展进行了综述，另一方面对以下内容进行了研究：用转基因和RNA干（RNAi）扰的方法建立不同FGF-2的表达量猕猴耳部皮肤细胞（MESF）系五组：过表达FGF-2（f1），过表达的阴性对照组（f2），FGF-2 RNA干扰组（f3），RNA干扰的阴性对照组（f4）以及未作任何处理的对照组（f5），这五组MESF的FGF-2表达量相对值为f1：f2：f3：f4：f5＝4：2：1：2：2；c-fos，TGF-β1，INHBA，Gremlin1在f1中表达量上升，在f3中表达量下降；BMP4，TGF-β2在f1中表达量下降，在f3中表达量上升；表明内源FGF-2能够作用于MESF的TGF-β信号通路，引起相关基因表达量的变化。用这些细胞作为饲养层分别培养两种ESC（猕猴ESC，R366. 4和兔ESC，RFESC） ，连续培养了10代，其中在f1上培养的两种ESC增殖速度都比对照组快，并且c-fos，TGF-β1，INHBA，Gremlin1，OCT-4，Nanog，Sox2表达量均上升，BMP4表达下调；在f3上培养的猕猴ESC增殖较慢，BMP4表达上调，c-fos，TGF-β1，INHBA，Gremlin1，OCT-4，Nanog，Sox2表达下调；f3上的兔ESC没有变化。表明ESC中的TGF-β信号通路也受到调节。五组猕猴和兔的ESC形成的EB均表达各胚层早期标记基因(marker)，但表达量有差异，f1上ESC形成的EB所有marker都低表达。实验结果表明饲养层中的FGF-2含量高低不仅影响自身相关基因的表达，还对ESC的增殖和维持自我更新有一定的作用。

兔胚胎干细胞分离建系及鉴定以及胚胎干细胞自我更新机制研究

人胚胎干细胞（ESC）的成功分离培养，吸引大批人对干细胞生物学的关注，特 别是ESC 在再生医学及人类早期胚胎发育研究的潜在价值。然而在人ESC 临床应用 之前需要找到合适的动物模型进行大量的预实验研究，从而评价其应用的安全性、有 效性及存活效率。因此，从其它物种建立稳定而可用的ESC 系也是必不可少的。ESC 能无限地自我更新并保持多潜能性，但控制其自我更新的分子机制现在仍然知之甚少 且物种间存在差异，了解ESC 的自我更新有利于提高建系率、改善培养体系及定向 分化体系。本文一方面对ESC 分离培养及自我更新机制的研究进展进行了综述，另 一方面对以下几个方面的内容进行了研究：1）建立了4 株稳定的兔ESC 系，能在体 外进行长期的培养并保持ESC 的多潜能Markers 及正常的XY 或XX 核型，具有碱性 磷酸酶活性、表达Oct-4、SSEA-1、SSEA-3、SSEA-4、TRA-1-60 及TRA-1-81。与人 和小鼠ESC 相似，兔ESC 表达多潜能基因（Oct-4、Nanog、Sox-2 及UTF-1），并表 达了与ESC 自我更新相关的信号通路（FGF、TGFβ及WNT）的许多基因。从形态 来说，兔ESC 与灵长类ESC 相似，但兔ESC 具有较快的增殖能力，与小鼠ESC 相类 似。在体外及体内兔ESC 均能分化成代表原始三胚层的各种细胞类型及组织。2) 从 受体抑制实验及生长因子的联合加入可以得出结论FGF 及TGF 信号通路对维持兔 ESC 的多潜能性发挥着重要的作用，这样的结果与人ESC 相类似。也表明FGF、TGF β及WNT 信号通路在兔ESC 的自我更新中都起着作用，而且他们之间可能形成了信 号调控网络，相互之间有着正负反馈作用。FGF2+Activin A 或TGFβ1+Noggin 的无 饲养层无血清培养体系不仅能显著抑制兔ESC 的分化，且能维持其长期的自我更新。 但与小鼠不同，TGFβ信号通路能影响其增殖能力，而对其多潜能性的维持并没有作 用。这就更说明了兔比小鼠更适宜成为人类疾病临床治疗之前的模型动物。3）四种 猕猴细胞系（MOF、MESF、MFG 和CMESF）可作为饲养层比MEFs（小鼠饲养层 细胞）更好或同等好支持猕猴ESC 的生长，保持其自我更新能力和分化的多能性。 而卵泡颗粒上皮样细胞（MFGE）不能支持猕猴ESC 的自我更新。进一步的研究表明 饲养层支持ESC 生长能力的差异可能是由于基因表达种类以及表达量上的差异而导 致的。

猕猴胚胎干细胞自我更新机理及WNT 信号通路在猕猴胚胎早期发育中作用的研究

尽管大部分动物实验是在啮齿类动物上开展的，我们仍然相信涉及人类的许多问题，如胚胎干细胞的体内功能、调亡和肿瘤形成等只有在非人灵长类模型上才能得到最好的回答。猕猴(标准的非人灵长类动物模型)在解剖、生理和代谢方面都和人类非常相似。人类很多神经疾病，如阿尔茨海默氏病、帕金森病，只能在非人灵长类模型上才能精确建模。所以研究猕猴胚胎干细胞自我更新的原理及猕猴胚胎早期发育，对研究免疫排斥，检测基于胚胎干细胞的治疗的可行性，安全性和有效性具有重要意义。本文一方面对胚胎干细胞维持自我更新和多潜能性的机理研究进行了综述，另一方面对以下两个方面的内容进行了研究： 1）运用寡核苷酸芯片和定量PCR 验证的方法来分析五株猕猴饲养层细胞的表达模式，期望发现在支持性和非支持性的饲养层细胞中差异性表达的基因。我们着重定位于饲养层胞外空间和细胞膜上的细胞因子，因为这些因子可以通过直接接触或通过膜结合受体激活下游信号通路，并最终促进猕猴胚胎干细胞的自我更新。我们发现在支持性的饲养层中有八个基因是高表达的，他们是GREM2， bFGF，KITLG，DKK3，GREM1，AREG，SERPINF1 和LTBP1； 经定量PCR 验证的SCF，bFGF 和GREM2 的表达情况都和芯片数据吻合。 2）为了描述在IVF (in vitro fertilized, 体外受精)，ICSI (intracytoplasmic sperm injection, 单精注射)，SCNT (somatic cell nuclear transfer, 体细胞核移植)和孤雌生殖猕猴囊胚中WNT 信号通路的表达情况，我们运用了信号通路特异性PCR Array 系统及免疫细胞化学来检测mRNA 和蛋白表达水平。其中，ICSI 作为IVF 胚胎的参照组，以排除显微操作对胚胎质量的影响。结果，我们发现非经典WNT/JNK 信号，而不是经典WNT 信号通路，在IVF 正常胚胎发育中起作用。而体细胞核移植和孤雌生殖的胚胎的WNT 信号通路基因表达明显高于正常胚胎。WNT 信号通路基因的表达模式可以作为胚胎质量的一个指示标准，有助于回答为什么猕猴 SCNT 和孤雌生殖胚胎发育异常。

C-Kit(+) Cells Isolated from Developing Kidneys Are a Novel Population of Stem Cells with Regenerative Potential

The presence of tissue specific precursor cells is an emerging concept in organ formation and tissue homeostasis. Several progenitors are described in the kidneys. However, their identity as a true stem cell remains elusive. Here, we identify a neonatal kidney-derived c-kit(+) cell population that fulfills all of the criteria as a stem cell. These cells were found in the thick ascending limb of Henle's loop and exhibited clonogenicity, self-renewal, and multipotentiality with differentiation capacity into mesoderm and ectoderm progeny. Additionally, c-kit(+) cells formed spheres in nonadherent conditions when plated at clonal density and expressed markers of stem cells, progenitors, and differentiated cells. Ex vivo expanded c-kit(+) cells integrated into several compartments of the kidney, including tubules, vessels, and glomeruli, and contributed to functional and morphological improvement of the kidney following acute ischemia-reperfusion injury in rats. Together, these findings document a novel neonatal rat kidney c-kit(+) stem cell population that can be isolated, expanded, cloned, differentiated, and used for kidney repair following acute kidney injury. These cells have important biological and therapeutic implications. STEM Cells 2013;31:1644-1656

c-Myc is required for maintenance of glioma cancer stem cells.

BACKGROUND: Malignant gliomas rank among the most lethal cancers. Gliomas display a striking cellular heterogeneity with a hierarchy of differentiation states. Recent studies support the existence of cancer stem cells in gliomas that are functionally defined by their capacity for extensive self-renewal and formation of secondary tumors that phenocopy the original tumors. As the c-Myc oncoprotein has recognized roles in normal stem cell biology, we hypothesized that c-Myc may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells. METHODOLOGY/PRINCIPAL FINDINGS: Based on previous methods that we and others have employed, tumor cell populations were enriched or depleted for cancer stem cells using the stem cell marker CD133 (Prominin-1). We characterized c-Myc expression in matched tumor cell populations using real time PCR, immunoblotting, immunofluorescence and flow cytometry. Here we report that c-Myc is highly expressed in glioma cancer stem cells relative to non-stem glioma cells. To interrogate the significance of c-Myc expression in glioma cancer stem cells, we targeted its expression using lentivirally transduced short hairpin RNA (shRNA). Knockdown of c-Myc in glioma cancer stem cells reduced proliferation with concomitant cell cycle arrest in the G(0)/G(1) phase and increased apoptosis. Non-stem glioma cells displayed limited dependence on c-Myc expression for survival and proliferation. Further, glioma cancer stem cells with decreased c-Myc levels failed to form neurospheres in vitro or tumors when xenotransplanted into the brains of immunocompromised mice. CONCLUSIONS/SIGNIFICANCE: These findings support a central role of c-Myc in regulating proliferation and survival of glioma cancer stem cells. Targeting core stem cell pathways may offer improved therapeutic approaches for advanced cancers.

Genetic Studies Identify Critical Biomarkers and Refine the Classification of Malignant Gliomas

Gliomagenesis is driven by a complex network of genetic alterations and while the glioma genome has been a focus of investigation for many years; critical gaps in our knowledge of this disease remain. The identification of novel molecular biomarkers remains a focus of the greater cancer community as a method to improve the consistency and accuracy of pathological diagnosis. In addition, novel molecular biomarkers are drastically needed for the identification of targets that may ultimately result in novel therapeutics aimed at improving glioma treatment. Through the identification of new biomarkers, laboratories will focus future studies on the molecular mechanisms that underlie glioma development. Here, we report a series of genomic analyses identifying novel molecular biomarkers in multiple histopathological subtypes of glioma and refine the classification of malignant gliomas. We have completed a large scale analysis of the WHO grade II-III astrocytoma exome and report frequent mutations in the chromatin modifier, alpha thalassemia mental retardation x-linked (ATRX), isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2), and mutations in tumor protein 53 (TP53) as the most frequent genetic mutations in low grade astrocytomas. Furthermore, by analyzing the status of recurrently mutated genes in 363 brain tumors, we establish that highly recurrent gene mutational signatures are an effective tool in stratifying homogeneous patient populations into distinct groups with varying outcomes, thereby capable of predicting prognosis. Next, we have established mutations in the promoter of telomerase reverse transcriptase (TERT) as a frequent genetic event in gliomas and in tissues with low rates of self renewal. We identify TERT promoter mutations as the most frequently mutated gene in primary glioblastoma. Additionally, we show that TERT promoter mutations in combination with IDH1 and IDH2 mutations are able to delineate distinct clinical tumor cohorts and are capable of predicting median overall survival more effectively than standard histopathological diagnosis alone. Taken together, these data advance our understanding of the genetic alterations that underlie the transformation of glial cells into neoplasms and we provide novel genetic biomarkers and multi – gene mutational signatures that can be utilized to refine the classification of malignant gliomas and provide opportunity for improved diagnosis.

Isolation of retinal progenitor and stem cells from the porcine eye

Purpose: Retinal progenitor cells (RPCs) and retinal stem cells (RSCs) from rodents and humans have been isolated and characterized in vitro. Transplantation experiments have confirmed their potential as tools for cell replacement in retinal degenerative diseases. The pig represents an ideal pre-clinical animal model to study the impact of transplantation because of the similarity of its eye to the human eye. However, little is known about porcine RPCs and RSCs. We aimed to identify and characterize in vitro RPCs and RSCs from porcine ocular tissues. Methods: Cells from different subregions of embryonic, postnatal and adult porcine eyes were grown in suspension sphere culture in serum-free medium containing basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Growth curves and BrdU incorporation assays were performed to establish the proliferative capacity of isolated porcine retina-derived RPCs and ciliary epithelium (CE)-derived RSCs. Self-renewal potential was investigated by subsphere formation assays. Changes in gene expression were assayed by reverse transcription polymerase chain reaction (RT-PCR) at different passages in culture. Finally, differentiation was induced by addition of serum to the cultures and expression of markers for retinal cell types was detected by immunohistochemical staining with specific antibodies. Results: Dissociated cells from embryonic retina and CE at different postnatal ages generated primary nestin- and Pax6-immunoreactive neurosphere colonies in vitro in numbers that decreased with age. Embryonic and postnatal retina-derived RPCs and young CE-derived RSCs displayed self-renewal capacity, generating secondary neurosphere colonies. However, their self-renewal and proliferation capacity gradually decreased and they became more committed to differentiated states with subsequent passages. The expansion capacity of RPCs and RSCs was higher when they were maintained in monolayer culture. Porcine RPCs and RSCs could be induced to differentiate in vitro to express markers of retinal neurons and glia. Conclusions: Porcine retina and CE contain RPCs and RSCs which are undifferentiated, self-renewing and multipotent and which show characteristics similar to their human counterparts. Therefore, the pig could be a useful source of cells to further investigate the cell biology of RPCs and RSCs and it could be used as a non-primate large animal model for pre-clinical studies on stem cell-based approaches to regenerative medicine in the retina.

Analysis of HSC activity and compensatory Hox gene expression profile in Hoxb cluster mutant fetal liver cells

Overexpression of Hoxb4 in bone marrow cells promotes expansion of hematopoietic stem cell (HSC) populations in vivo and in vitro, indicating that this homeoprotein can activate the genetic program that determines self-renewal. However, this function cannot be solely attributed to Hoxb4 because Hoxb4(-/-) mice are viable and have an apparently normal HSC number. Quantitative polymerase chain reaction analysis showed that Hoxb4(-/-) c-Kit(+) fetal liver cells expressed moderately higher levels of several Hoxb cluster genes than control cells, raising the possibility that normal HSC activity in Hoxb4(-/-) mice is due to a compensatory up-regulation of other Hoxb genes. In this study, we investigated the competitive repopulation potential of HSCs lacking Hoxb4 alone, or in conjunction with 8 other Hoxb genes. Our results show that Hoxb4(-/-) and Hoxb1-b9(-/-) fetal liver cells retain full competitive repopulation potential and the ability to regenerate all myeloid and lymphoid lineages. Quantitative Hox gene expression profiling in purified c-KIt(+) Hoxb1-bg(-/-) fetal liver cells revealed an interaction between the Hoxa, b, and c clusters with variation in expression levels of Hoxa4, -a11, and -c4. Together, these studies show a complex network of genetic interactions between several Hox genes in primitive hematopoietic cells and demonstrate that HSCs lacking up to 30% of the active Hox genes remain fully competent.