"Reprogram Enablement" as an Assay for Identifying Early Oncogenic Pathways by Their Ability to Allow Neoplastic Cells to Reacquire an Epiblast State.

One approach to understanding how tissue-specific cancers emerge is to determine the requirements for "reprograming" such neoplastic cells back to their developmentally normal primordial pre-malignant epiblast-like pluripotent state and then scrutinizing their spontaneous reconversion to a neoplasm, perhaps rendering salient the earliest pivotal oncogenic pathway(s) (before other aberrations accumulate in the adult tumor). For the prototypical malignancy anaplastic thyroid carcinoma (ATC), we found that tonic RAS reduction was obligatory for reprogramming cancer cells to a normal epiblast-emulating cells, confirmed by changes in their transcriptomic and epigenetic profiles, loss of neoplastic behavior, and ability to derive normal somatic cells from their "epiblast organoids." Without such suppression, ATCs re-emerged from the clones. Hence, for ATC, RAS inhibition was its "reprogram enablement" (RE) factor. Each cancer likely has its own RE factor; identifying it may illuminate pre-malignant risk markers, better classifications, therapeutic targets, and tissue-specification of a previously pluripotent, now neoplastic, cell.

Teratogenic Rubella Virus Alters the Endodermal Differentiation Capacity of Human Induced Pluripotent Stem Cells.

The study of congenital virus infections in humans requires suitable ex vivo platforms for the species-specific events during embryonal development. A prominent example for these infections is rubella virus (RV) which most commonly leads to defects in ear, heart, and eye development. We applied teratogenic RV to human induced pluripotent stem cells (iPSCs) followed by differentiation into cells of the three embryonic lineages (ecto-, meso-, and endoderm) as a cell culture model for blastocyst- and gastrulation-like stages. In the presence of RV, lineage-specific differentiation markers were expressed, indicating that lineage identity was maintained. However, portrait analysis of the transcriptomic expression signatures of all samples revealed that mock- and RV-infected endodermal cells were less related to each other than their ecto- and mesodermal counterparts. Markers for definitive endoderm were increased during RV infection. Profound alterations of the epigenetic landscape including the expression level of components of the chromatin remodeling complexes and an induction of type III interferons were found, especially after endodermal differentiation of RV-infected iPSCs. Moreover, the eye field transcription factors RAX and SIX3 and components of the gene set vasculogenesis were identified as dysregulated transcripts. Although iPSC morphology was maintained, the formation of embryoid bodies as three-dimensional cell aggregates and as such cellular adhesion capacity was impaired during RV infection. The correlation of the molecular alterations induced by RV during differentiation of iPSCs with the clinical signs of congenital rubella syndrome suggests mechanisms of viral impairment of human development.

Drug-based modulation of endogenous stem cells promotes functional remyelination in vivo.

Multiple sclerosis involves an aberrant autoimmune response and progressive failure of remyelination in the central nervous system. Prevention of neural degeneration and subsequent disability requires remyelination through the generation of new oligodendrocytes, but current treatments exclusively target the immune system. Oligodendrocyte progenitor cells are stem cells in the central nervous system and the principal source of myelinating oligodendrocytes. These cells are abundant in demyelinated regions of patients with multiple sclerosis, yet fail to differentiate, thereby representing a cellular target for pharmacological intervention. To discover therapeutic compounds for enhancing myelination from endogenous oligodendrocyte progenitor cells, we screened a library of bioactive small molecules on mouse pluripotent epiblast stem-cell-derived oligodendrocyte progenitor cells. Here we show seven drugs function at nanomolar doses selectively to enhance the generation of mature oligodendrocytes from progenitor cells in vitro. Two drugs, miconazole and clobetasol, are effective in promoting precocious myelination in organotypic cerebellar slice cultures, and in vivo in early postnatal mouse pups. Systemic delivery of each of the two drugs significantly increases the number of new oligodendrocytes and enhances remyelination in a lysolecithin-induced mouse model of focal demyelination. Administering each of the two drugs at the peak of disease in an experimental autoimmune encephalomyelitis mouse model of chronic progressive multiple sclerosis results in striking reversal of disease severity. Immune response assays show that miconazole functions directly as a remyelinating drug with no effect on the immune system, whereas clobetasol is a potent immunosuppressant as well as a remyelinating agent. Mechanistic studies show that miconazole and clobetasol function in oligodendrocyte progenitor cells through mitogen-activated protein kinase and glucocorticoid receptor signalling, respectively. Furthermore, both drugs enhance the generation of human oligodendrocytes from human oligodendrocyte progenitor cells in vitro. Collectively, our results provide a rationale for testing miconazole and clobetasol, or structurally modified derivatives, to enhance remyelination in patients.

Evaluation of the cellular origins of heterotopic ossification.

Heterotopic ossification (HO), acquired or hereditary, is featured by the formation of bone outside of the normal skeleton. Typical acquired HO is a common, debilitating condition associated with traumatic events. Cardiovascular calcification, an atypical form of acquired HO, is prevalent and associated with high rates of cardiovascular mortality. Hereditary HO syndromes, such as fibrodysplasia ossificans progressiva and progressive osseous heteroplasia, are rare, progressive, life-threatening disorders. The cellular origins of HO remain elusive. Some bona fide contributing cell populations have been found through genetic lineage tracing and other experiments in vivo, and various other candidate populations have been proposed. Nevertheless, because of the difficulties in establishing cellular phenotypes in vivo and other confounding factors, the true identities of these populations are still uncertain. This review critically evaluates the accumulating data in the field. The major focus is on the candidate populations that may give rise to osteochondrogenic lineage cells directly, not the populations that may contribute to HO indirectly. This issue is important not solely because of the clinical implications, but also because it highlights the basic biological processes that govern bone formation.

Fine structure of progenitor cells in early ectopic human embryos.

This study has documented the major types of lineage progenitor cells at the second level of cell differentiation after the establishment of the primary germ layers in ectopic human embryos in vivo. These correspond to stages 8 and 9 of embryogenesis (weeks 3-4) in the Carnegie collection. The aim of this study was to provide images of fine structure of tissue progenitor cells to compare them with current imaging of their equivalent stem cells identified using fluorescent stem cell markers. These include neural, mesenchymal, endodermal, ectodermal (epidermal) and haematopoietic progenitor cells, including those for amniotic, yolk sac and chorionic tissues that are used in current stem cell research. Neural induction by the notochord has been imaged. This study should give valuable clues to understand the pattern of cell differentiation of embryonic stem cells (ESC) in vitro, which are more or less mimicked in ESC colonies, embryoid bodies and neurospheres as documented in the literature. The fine structure of week-3 and week-4 human ectopic embryos is presented to demonstrate progenitor tissue cells that will eventually form the brain, spinal cord, skin, gut, heart, blood, muscle, bone and other tissues of the human body later on in development. These images should help stem cell researchers using fluorescent markers and other techniques to identify embryonic and adult stem cells in culture.

Human-induced pluripotent stem cells produced under xeno-free conditions.

Induced pluripotent stem cells (iPSCs) have radically advanced the field of regenerative medicine by making possible the production of patient-specific pluripotent stem cells from adult individuals. While cell differentiation protocols have been successfully developed, and animal models of human disease have proved that these cells have the potential to treat human diseases and conditions produced as a consequence of aging, degeneration, injury, and birth defects, logistical issues still remain unsolved and hamper the possibility of testing these cells in human clinical trials. Among them is the widely spread use of animal products for the generation and culture of iPSCs. We report here a xeno-free iPSC generation system that addresses all the steps of iPSCs production including the isolation and culture of adult skin fibroblasts, and iPSCs generation, expansion, and maintenance. iPSCs generated with a polycistronic lentiviral vector under xeno-free conditions displayed markers of pluripotency and gave rise to embryoid bodies (EBs) displaying indicators of the 3 primary germ layers. Xeno-free iPSCs injected into nude mice produced classic teratomas, and teratoma explants cultured under conditions favoring fibroblastic cells gave rise to cells morphologically indistinguishable from input cells. Protocols here described will facilitate the implementation of new cellular therapies for preclinical and clinical studies, potentially reducing the regulatory burden without compromising the differentiation potential of the cells.

Germinal matrix hemorrhage of prematurity: treatment approaches and outcomes in a single institutional review in the Ukraine.

OBJECTIVE: To review the treatment outcome of germinal matrix hemorrhage (GMH) in premature infants in a single Ukrainian institution in an effort to determine optimal diagnostic and therapeutic approaches. MATERIALS AND METHODS: Eight hundred and thirty-five premature newborns (gestational age 33.0 +/- 2.50 weeks, birth weight 2,124.81 +/- 282.54 g; mean +/- SD) were examined for the development of perinatal hypoxic-ischemia injury and asphyxia condition. This research focuses on various types of massive intracranial hemorrhage (ICH) and posthemorrhagic hydrocephalus (PHH). The diagnostic methods were based on intracranial imaging studies and clinical features that are present at birth. The therapeutic and preventive strategies consist of parental counseling, supportive and rehabilitative care for affected infants. Surgical intervention was indicated for the prevention and treatment of severe PHH. Thirty-four preterm infants were treated by ventricular-peritoneal/subgaleal shunting with close monitoring of intracerebral pressure. RESULTS: Massive GMH took place at 32-35 gestational weeks as a result of increased periventricular anastomosis. GMH was unusual in full-term newborns, whereas cerebral hypoxic-ischemic injuries were more common in full-term neonates. Approximately 98% of premature infants with low birth weight survived and 2% died due to respiratory distress syndrome and other complications. ICH which occurs in neonates at 24-28 gestational weeks was mainly due to immature vascular walls and insufficiency of vascular anastomosis at the germinal matrix. CONCLUSIONS: ICH occurring in the germinal matrix of premature newborns is closely related to the development of the brain vasculature. Evacuation of the hematoma is more detrimental than beneficial, despite the rapid strides being taken to keep low-birth weight premature infants alive. Therefore, the treatment of ICH and PHH requires a fundamental understanding of pathogenetic changes, which is necessary for the neurorehabilitation and immediate elimination of cerebral compression and its complications.

Brain and eye malformations resembling Walker-Warburg syndrome are recapitulated in mice by dystroglycan deletion in the epiblast.

Walker-Warburg syndrome (WWS) is a severe congenital disease that is characterized by brain and eye malformations and lethality during the first year of life. Genetic mutations have been identified in a subset of WWS patients, but a majority of clinical cases have unknown etiologies. POMT1 and POMT2, two of the causative genes, form an active enzyme complex in the posttranslational biosynthetic pathway of dystroglycan. Deletion of either Pomt1 or the dystroglycan gene causes early embryonic lethality in mice. Here we report that mice with epiblast-specific loss of dystroglycan develop brain and eye defects that broadly resemble the clinical spectrum of the human disease, including aberrant neuron migration, hydrocephalus, and malformations of the anterior and posterior chambers of the eye. Breaches of basement membranes coincide with the pathology, revealing an important function for dystroglycan in the morphogenesis of the brain and eye. These findings demonstrate the central role of dystroglycan in WWS and suggest that novel defects in posttranslational processing or mutations of the dystroglycan gene itself may underlie cases in which no causative mutation has been found.

Assessment of drug induced developmental toxicity using human embryonic stem cells.

Embryonic stem (ES) cells are unique as they have the potential to be generated in large numbers and the ability to differentiate into the three germ layers via embryoid body (EB) formation. This property could be utilized as an index to study initial mammalian development. We have investigated the utility of a comprehensively characterized human ES (hES) cell line (ReliCellhES1) for testing the embryotoxic effects of compounds using cytotoxicity assays. Further, we performed real time gene expression analysis to check the alterations in germ layer markers expression upon drug treatment. The results show that assays using hES cells could serve as a reliable, sensitive and robust method to assess embryotoxic potential of compounds. They also provide a proof of concept that hES cells can be used as an in vitro model to demonstrate developmental toxicity, and to examine the germ layer-specific effects on differentiating EBs.

Epiblastic Cited2 deficiency results in cardiac phenotypic heterogeneity and provides a mechanism for haploinsufficiency.

AIMS: Deletion of the transcription factor Cited2 causes penetrant and phenotypically heterogenous cardiovascular and laterality defects and adrenal agenesis. Heterozygous human CITED2 mutation is associated with congenital heart disease, suggesting haploinsufficiency. Cited2 functions partly via a Nodal-->Pitx2c pathway controlling left-right patterning. In this present study we investigated the primary site of Cited2 function and mechanisms of haploinsufficiency. METHODS AND RESULTS: A Cited2 conditional allele enabled its deletion in particular cell lineages in mouse development. A lacZ reporter cassette allowed indication of deletion. Congenic Cited2 heterozygous mice were used to investigate haploinsufficiency. Embryos were examined by magnetic resonance imaging, by sectioning and by quantitative real-time polymerase chain reaction (qRT-PCR). Epiblast-specific deletion of Cited2 using Sox2Cre recapitulated penetrant and phenotypically heterogenous cardiovascular and laterality defects. Neural crest-specific deletion using Wnt1Cre affected cranial ganglia but not cardiac development. Mesodermal deletion with Mesp1Cre resulted in low penetrance of septal defect. Mesodermal deletion with T-Cre resulted in adrenal agenesis, but infrequent cardiac septal and laterality defects. beta-Galatactosidase staining and qRT-PCR demonstrated the efficiency and location of Cited2 deletion. Murine Cited2 heterozygosity is itself associated with cardiac malformation, with three of 45 embryos showing ventricular septal defect. Cited2 gene expression in E13.5 hearts was reduced 2.13-fold in Cited2(+/-) compared with wild-type (P = 2.62 x 10(-6)). The Cited2 target gene Pitx2c was reduced 1.5-fold in Cited2(+/-) (P = 0.038) hearts compared with wild-type, and reduced 4.9-fold in Cited2(-/-) hearts (P = 0.00031). Pitx2c levels were reduced two-fold (P = 0.009) in Cited2(+/-) embryos, in comparison with wild-type. Cited2 and Pitx2c expression were strongly correlated in wild-type and Cited2(+/-) hearts (Pearson rank correlation = 0.68, P = 0.0009). Cited2 expression was reduced 7474-fold in Sox2Cre deleted hearts compared with controls (P = 0.00017) and Pitx2c was reduced 3.1-fold (P = 0.013). Deletion of Cited2 with Mesp1Cre resulted in a 130-fold reduction in cardiac Cited2 expression compared with control (P = 0.0002), but Pitx2c expression was not affected. CONCLUSION: These results indicate that phenotypically heterogenous and penetrant cardiac malformations in Cited2 deficiency arise from a primary requirement in epiblast derivatives for left-right patterning, with a secondary cell-autonomous role in the mesoderm. Cardiac malformation associated with Cited2 haploinsufficiency may occur by reducing expression of key Cited2 targets such as Pitx2c.

Adult intramedullary mature teratoma of the spinal cord: review of the literature illustrated with an unusual example.

Intramedullary mature teratomas particularly in adults are rarely encountered. In this manuscript the authors have reviewed the adult intramedullary lesions of the spinal cord published in the literature that are harbouring the characteristics of a mature teratoma and analysed the results with respect to histopathology, epidemiology, diagnostic methods and treatment. An illustrative case of an extremely unusual localization is also presented.

Degenerative changes and cell death in long-living homo- and heterotopic transplants from embryonic germ layers of rat neocortex.

Morphological study of allotransplants of rat embryonic neocortex 14-18 months after transplantation into the neocortex, lateral cerebral ventricle, and sciatic nerve of adult animals revealed death of nerve and glial cells in the delayed postoperation period independently on the site of transplantation. After heterotopic transplantation the count of degenerated neurons was 2 times higher that after homotopic transplantation. In heterotopic transplants a considerable number of grafted neurons underwent reversible and irreversible degenerative changes accompanied by their premature aging. Neuronal death is probably determined by insufficiency of trophic influence from afferent structures and target tissues. We hypothesized that antiapoptotic preparations can be used for prevention of transplanted cell death. It was also found that degeneration of neurons was associated with impaired vascularization of transplants and pronounced immune reaction of the recipient in late posttransplantation period. Transplantation of embryonic brain structures can serve as a model system in studies concerning involutive and pathological processes in the central nervous system and in the search for factors improving survival of neurons.

Transdifferentiation and nuclear reprogramming in hematopoietic development and neoplasia.

Cell transplantation and tissue regeneration studies indicate a surprisingly broad developmental potential for lineage-committed hematopoietic stem cells (HSCs). Under these conditions HSCs transition into myocytes, neurons, hepatocytes or other types of nonhematopoietic effector cells. Equally impressive is the progression of committed neuronal stem cells (NSCs) to functional blood elements. Although critical cell-of-origin issues remain unresolved, the possibility of lineage switching is strengthened by a few well-controlled examples of cell-type conversion. At the molecular level, switching probably initiates from environmental signals that induce epigenetic modifications, resulting in changes in chromatin configuration. In turn, these changes affect patterns of gene expression that mediate divergent developmental programs. This review examines recent findings in nuclear reprogramming and cell fusion as potential causative mechanisms for transdifferentiation during normal and malignant hematopoiesis.

Clinicopathological study of seven cases of spinal cord teratoma: a possible germ cell origin.

AIMS: To establish the clinical and pathological aspects of teratoma affecting the spinal cord. METHOD AND RESULTS: We reviewed our neurosurgical records for the last 15 years and found seven cases of teratoma of the spinal cord. The cases were reviewed clinically, radiologically and pathologically using immunohistochemical markers to identify various tissue components. We found that spinal cord teratoma is an extremely rare tumour of spinal cord affecting patients aged 23-47 years and of approximately equal male to female distribution. The terminal portions of spinal cord and intradural location of the tumour are common. Three cases were associated with vertebral anomaly. Most tumours showed benign (mature) components derived from more than one germ cell layer; one showed malignant adenocarcinomatous component. All cases were treated by surgical resection and two recurred at 6 and 10 year intervals without malignant transformation. CONCLUSION: Spinal cord teratoma is a rare, mainly benign tumour, which could be associated with vertebral anomaly. The pathogenesis of this tumour is controversial, possibly due to germinal cell aberration.

Prenatal pseudocysts of the germinal matrix in preterm infants.

Sonographic characteristics of germinal-matrix (PGM) pseudocysts of prenatal origin detected on cranial ultrasound in preterm newborns were correlated with their outcomes. PGM cysts were classified as typical or atypical, according to their location. Typical PGM cysts were present at the head of the caudate nucleus or slightly medially, adjacent to the foramen of Monro. Cysts were defined as atypical when they were located subependymally elsewhere. Only one infant of 16 with a typical PGM cyst presented with psychomotor retardation. His cerebral scan also showed subependymal calcifications due to cytomegalovirus infection. Three babies had cysts in the frontal periventricular zones (atypical PGM cyst). They had negative cranial MRI (12-15 months of age) and normal neurological follow-up (24 months). In conclusion, isolated prenatal PGM cysts in preterm infants correlate with a normal outcome.

Precision of ultrasound diagnosis of pathologically verified lesions in the brains of very preterm infants.

Abnormalities detected by a mechanical sector scanner were compared 'blind' with autopsy findings in the brains of 56 infants born at less than 33 weeks gestation. Intraventricular haemorrhage was found in 53 of 112 hemispheres and had been accurately diagnosed by ultrasound (sensitivity 91 per cent; specificity 81 per cent). Isolated germinal layer haemorrhage was less successfully identified (sensitivity 61 per cent; specificity 78 per cent); false-positive diagnoses were partly due to difficulty in distinguishing haemorrhage from the normal choroid plexus in extremely preterm infants. Haemorrhagic parenchymal lesions were correctly identified in nine infants (sensitivity 82 per cent; specificity 97 per cent). Only 11 of 39 hemispheres with histological evidence of hypoxic-ischaemic injury, without marked bleeding, were correctly identified by ultrasound (sensitivity 28 per cent), mainly because of failure to detect small areas of periventricular leucomalacia and diffuse gliosis. 10 hemispheres with periventricular echodensities thought to represent leucomalacia showed no histological evidence of hypoxic-ischaemic injury (specificity 86 per cent). Ventricular dilatation in seven infants was always associated with evidence of hypoxic-ischaemic injury at autopsy.

Gestational changes in the germinal matrix of the normal rhesus monkey fetus.

To explain the reported predisposition to germinal matrix hemorrhage in premature infants, pathogenetically important morphological features of the germinal matrix should be present in the 3rd trimester and rapidly change near term. Such features were sought in this study of the germinal matrix and its vasculature in normal rhesus monkey fetuses. The matrix cells, glia, ependyma, and capillaries showed no important structural changes during the 3rd trimester. The terminal vein tributaries were greatly enlarged by 148 days, but cellular and collagen support in their walls was minimal at this time. The latter features developed by the final days of gestation. These findings do not support a structural immaturity or specialization of the germinal matrix predisposing to germinal matrix hemorrhage. Our results, therefore, support the recent emphasis on physiological parameters in the pathogenesis and prevention of germinal matrix hemorrhage.

Immunomorphologic changes in regional lymph nodes associated with cancer.

Morphologic patterns in regional lymph nodes draining resected tumors correlate with in vitro immunologic data and have been reported to predict survival, stage of disease, and presence or absence of metastases. The strength of the relationship between immunomorphologic findings and survival rates varies with the type of tumor, perhaps because of relative immunogenicity, tumor size and growth rate, or even the stimulation of the lymph nodes by infection. Nevertheless, there is remarkable agreement among investigators on key observations, which yield more prognostic information than can be obtained by standard methods.

A systemic lymphoproliferative disorder with morphologic features of Castleman's disease. Pathological findings in 15 patients.

This report describes the nodal and extranodal lesions observed in 15 patients with a generalized disorder that had been histologically diagnosed as Castleman's disease. The disorder was characterized by severe constitutional symptoms, constant involvement of multiple peripheral lymph nodes, and frequent hepatosplenomegaly, in association with clinical and laboratory features reminiscent of a "collagen disease." The clinical course was chronic, with remissions and exacerbations in seven patients, and aggressive and fatal in eight. The material examined included multiple lymph node biopsies, four surgical specimens of spleen, one open lung biopsy, and material from four autopsies. The diagnostic morphological findings were observed in the nodes and were represented by the following histologic triad: diffuse marked plasmacytosis, from the medulla to the subcapsular areas; prominence of the germinal centers; and good preservation of the architecture. One variant of this basic pattern featured abundant immunoblasts and blood vessels. The process appears to be a systemic reactive proliferation of B-lymphocytes, perhaps resulting from faulty immune regulation. Morphologic similarities indicate a relationship between this multicentric disorder and Castleman's disease of plasmacellular type. However, there are distinct differences between them in clinical presentation and evolution, and, consequently, in therapeutic approach.