"Miniguts" from plucked human hair meet Crohn's disease.

Human pluripotent stem cells represent a powerful tool to study human embryonic development and disease but also open up novel strategies for cell replacement therapies. Their capacity to give rise to every cell type of the human body, meanwhile, enables researchers to generate high yields of mesodermal, ectodermal, but also endodermal-derived tissues such as hepatic, pancreatic, or intestinal cells. Another progress in the field came with the advent of 3-dimensional culture conditions, so-called organoids, which facilitate maturation of stem cells and in turn more faithfully recapitulate human tissue architecture. While several studies reported the derivation of organoid cultures from adult intestinal tissue, the derivation of intestinal organoids derived from plucked human hair of Crohn's disease patients has not been reported. The current research project reports such successful generation and characterization of induced pluripotent stem cells (iPSCs) derived from hair sheet keratinocyte cultures of a patient with Crohn's disease. Stepwise differentiation along the intestinal lineage showed no differences in intermediate stages such as definitive endoderm formation. We also directed the patterned primitive gut tube toward intestinal organoids resembling the cellular architecture of human "miniguts". As expected from current pathophysiological knowledge on Crohn's disease, there were no obvious morphological differences in the "miniguts" derived from healthy control and diseased patient-induced pluripotent stem cells. Taken together, our platform will enable for detailed and complementary phenotyping of the pathophysiology of Crohn's disease in a novel disease-in-a-dish format.

Pcgf6, a polycomb group protein, regulates mesodermal lineage differentiation in murine ESCs and functions in iPS reprogramming.

Polycomb group (PcG) proteins comprise evolutionary conserved factors with essential functions for embryonic development and adult stem cells. PcG proteins constitute two main multiprotein polycomb repressive complexes (PRC1 and PRC2) that operate in a hierarchical manner to silence gene transcription. Functionally distinct PRC1 complexes are defined by Polycomb group RING finger protein (Pcgf) paralogs. So far, six Pcgf paralogs (Pcgf1-6) have been identified as defining components of different PCR1-type complexes. Paralog-specific functions are not well understood. Here, we show that Pcgf6 is the only Pcgf paralog with high expression in undifferentiated embryonic stem cells (ESCs). Upon differentiation Pcgf6 expression declines. Following Pcgf6 kockdown (KD) in ESCs, the expression of pluripotency genes decreased, while mesodermal- and spermatogenesis-specific genes were derepressed. Concomitantly with the elevated expression of mesodermal lineage markers, Pcgf6 KD ESCs showed increased hemangioblastic and hematopoietic activities upon differentiation suggesting a function of Pcgf6 in repressing mesodermal-specific lineage genes. Consistant with a role in pluripotency, Pcgf6 replaced Sox2 in the generation of germline-competent induced pluripotent stem (iPS) cells. Furthermore, Pcgf6 KD in mouse embryonic fibroblasts reduced the formation of ESC-like colonies in OSKM-driven reprogramming. Together, these analyses indicate that Pcgf6 is nonredundantly involved in maintaining the pluripotent nature of ESCs and it functions in iPS reprogramming.

A conserved C-terminal sequence that is deleted in v-ErbA is essential for the biological activities of c-ErbA (the thyroid hormone receptor).

The thyroid hormone (T3) receptor type alpha, the c-ErbA alpha proto-oncoprotein, stimulates transcription of T3-dependent promoters, interferes with AP-1 activity, and induces erythroid differentiation in a ligand-dependent manner. The v-ErbA oncoprotein does not bind hormone and has lost all of these activities. Using c-ErbA/v-ErbA chimeras, we found that a deletion of 9 amino acids, conserved among many members of the nuclear receptor superfamily, which are located at the extreme carboxy terminus of c-ErbA alpha is responsible for loss of both transactivation and transcriptional interference activities. Single, double, and triple amino acid substitutions within this region completely abolished T3-dependent transcriptional activation, interference with AP-1 activity, and decreased T3 binding by c-ErbA alpha. However, the lower T3 binding by these mutants does not fully account for the loss of transactivation and transcriptional interference, since a c-ErbA/v-ErbA chimera which was similarly reduced in T3 binding activity has retained both of these functions. Deletion of homologous residues in the retinoic acid receptor alpha (RAR alpha) resulted in a similar loss of transactivation and transcriptional interference activities. The ability of c-ErbA alpha to induce differentiation of transformed erythroblasts is also impaired by all of the mutations introduced into the conserved carboxy-terminal sequence. We conclude that this 9-amino-acid conserved region is essential for normal biological function of c-ErbA alpha and RAR alpha and possibly other T3 and RA receptors.

Specific protein binding to the simian virus 40 enhancer in vitro.

HeLa cell nuclear extracts and wild-type or mutated simian virus 40 enhancer DNA were used in DNase I footprinting experiments to study the interaction of putative trans-acting factors with the multiple enhancer motifs. We show that these nuclear extracts contain proteins that bind to these motifs. Because point mutations which are detrimental to the activity of a particular enhancer motif in vivo specifically prevent protection of that motif against DNase I digestion in vivo, we suggest that the bound proteins correspond to trans-acting factors involved in enhancement of transcription. Using mutants in which the two domains A and B of the simian virus 40 enhancer are either separated by insertion of DNA fragments or inverted with respect to their natural orientation, we also demonstrate that the trans-acting factors bind independently to the two domains.

Two different mRNAs are transcribed from a single genomic locus encoding the chicken erythrocyte anion transport proteins (band 3).

The chicken erythrocyte anion transport protein (band 3 of the erythrocyte cytoskeleton) is a central component taking part in two widely divergent functions of erythroid cells; it is a primary determinant of cytoskeletal architecture and responsible for electroneutral Cl-/HCO3- exchange across the plasma membrane. To analyze interesting aspects of the developmental regulation of this gene, we have cloned the cDNA and genomic counterparts of the erythroid-specific anion transport protein. We show that a single genetic locus for band 3 encodes two different erythroid cell-specific mRNAs, with different translational initiation sites, which predict polypeptides of sizes very close to those observed in vivo. In vitro translation and immune precipitation of synthetic mRNA derived from one putative fully encoding cDNA clone demonstrate that this clone gives rise to a protein which is identical in size and antigenicity to bona fide chicken erythroid band 3.

Molecular switches and developmental potential of adult stem cells.

Stem cell commitment and differentiation entails the successive loss of self-renewal and developmental potential, and results in the final restriction to a terminally differentiated mature cell type. Hematopoiesis, the development of blood cells from hematopoietic stem cells in bone marrow, is particularly well studied, and at different branching points within the hematopoietic system multiple developmental intermediates have been identified. Here we describe a Flt3+ CD11b+ multipotent progenitor that can be amplified in vitro by a specific cytokine combination to high cell numbers, and following adoptive transfer into syngeneic mice, it generates dendritic cells but also additional mature cell types. By employing gene expression profiling with DNA microarrays and knockout mouse models, we demonstrate that the helix-loop-helix (HLH) transcription factor Id2 (inhibitor of DNA binding/differentiation 2) acts as a molecular switch in development of Langerhans cells (LCs), the cutaneous contingent of dendritic cells (DCs), and of specific DC subsets and B cells.

Modulation of normal erythroid differentiation by the endogenous thyroid hormone and retinoic acid receptors: a possible target for v-erbA oncogene action.

The v-erbA oncogene, a mutated version of the thyroid hormone receptor alpha (c-erbA/TR-alpha), inhibits erythroid differentiation and constitutively represses transcription of certain erythrocyte genes, suggesting a normal function of the proto-oncogene c-erbA in erythropoiesis. Here we demonstrate that the endogenous thyroid hormone receptor alpha (c-erbA/TR-alpha) and the closely related retinoic acid receptor alpha (RAR-alpha) play a role in the regulation of normal erythroid differentiation. Retinoic acid (RA) distinctly modulated the erythroid differentiation program of normal erythroid progenitors and erythroblasts reversibly transformed by a conditional tyrosine kinase oncogene. When added pulsewise to immature cells, differentiation was accelerated while more mature cells underwent premature cell death. Thyroid hormone (T3) alone caused similar but weaker effects. Interestingly, T3 strongly enhanced the action of RA, suggesting cooperative action of the two receptors in modulating erythroid differentiation. Expression of the human RAR-alpha in receptor-negative erythroblasts conferred RA-induced regulation of differentiation to the otherwise unresponsive cells, thus showing that the RAR-alpha is essential for the RA effect. Likewise, enhanced expression of exogenous c-erbA/TR-alpha in erythroblasts rendered them susceptible to modulation of differentiation by T3, suggesting a similar function of both receptors.

The fibroblast growth factor receptor FGFR-4 acts as a ligand dependent modulator of erythroid cell proliferation.

Receptor and non-receptor tyrosine kinases constitute a large family of proteins that play a pivotal role in hematopoiesis. Here we conducted a comprehensive survey of tyrosine kinase gene expression in primary erythroid progenitor cells from bone marrow by employing a PCR-based strategy that targets the conserved kinase encoding region. We demonstrate that erythroid progenitor cells express several receptor and non-receptor tyrosine kinases, like c-kit, Jak1, Ryk, FAK, Syk, Arg, Csk and members of the insulin receptor family. Specific changes in the expression profile of tyrosine kinases were observed following differentiation induction. We also report on the identification of a new ligand dependent modulator of erythropoiesis, fibroblast growth factor receptor-4 (FGFR-4). FGFR-4 is effectively expressed in erythroid progenitors and downregulated when cells differentiate. Furthermore, the FGFR-4 ligand, basic fibroblast growth factor (bFGF), enhanced erythroid cell proliferation induced by SCF or insulin, and thus modulated both erythroid proliferation and differentiation in vitro.

The Myb leucine zipper is essential for leukemogenicity of the v-Myb protein.

The AMV v-Myb oncoprotein causes oncogenic transformation of myelomonocytic cells in vivo and in vitro. Its transforming capacity is strictly dependent upon the N-terminal DNA binding domain, the central transactivation region, and on the C-terminal domain containing a putative leucine zipper motif. Here we show that the v-MybL3,4A mutant, in which Leu325 and Leu332 of the leucine zipper have been replaced by alanines, failed to induce leukemia in virus infected chicken. This demonstrates that the leucine zipper domain is indispensable for v-myb induced leukemogenesis in vivo. v-MybL3,4A was, however, still able to transform myelomonocytic cells from chicken bone marrow in vitro. Yet, while v-mybL3,4A transformed cells were impaired in growth at 37 degrees C, they failed to grow at 42 degrees C, the physiological body temperature of avian species. This might explain the loss of v-MybL3,4A leukemogenic potential in vivo. We also demonstrate that the v-Myb leucine zipper domain interacts in vitro with two host cell proteins, p26 and p28. This interaction is compromised in v-MybL3,4A indicating that binding of v-Myb to p26 and p28 might be important for the leukemogenic potential of v-Myb.

Retinoid X receptor and c-cerbA/thyroid hormone receptor regulate erythroid cell growth and differentiation.

Nuclear receptors are important regulators of erythroid cell development. Here we investigated the impact of retinoid X receptor (RXR), retinoic acid receptor (RAR), and of the c-erbA/thyroid hormone (T3) receptor (c-erbA/TR) on growth and differentiation of erythroid cells using an in vitro culture system of stem cell factor-dependent erythroid progenitors. RXR, RAR, and c-erbA/TR-specific ligands were found to induce erythroid-specific gene expression and to accelerate erythroid differentiation in culture, with T3 being most effective. Furthermore, while ligand-activated c-erbA/TR accelerated differentiation, unliganded c-erbA/TR effectively blocked differentiation and supported sustained progenitor growth in culture. Thus, c-erbA/TR appears to act as a binary switch affecting erythroid cell fate: unliganded c-erbA/TR supports growth while ligand-activated c-erbA/TR induces differentiation. Additionally, to determine the impact of RXR for erythroid cell development, dominant interfering mutant RXRs, lacking the transcriptional activator functions AF-1 and AF-2, or AF-2 only, or the entire DNA-binding domain, were introduced into erythroid progenitor cells via recombinant retrovirus vectors and analyzed for RXR-specific effects. It was found that expression of wild-type RXR and of the RXR mutants devoid of AF-1 and/or AF-2 supported a transient outgrowth of erythroid cells. In marked contrast, expression of the dominant interfering deltaDNA-binding domain RXR, containing a deletion of the entire DNA-binding domain, was incompatible with erythroid cell growth in vitro, suggesting a pivotal role of RXR for erythroid cell development.

Efficient gene delivery into human dendritic cells by adenovirus polyethylenimine and mannose polyethylenimine transfection.

Gene-modified human dendritic cells (DCs) were generated by transfection with adenovirus polyethylenimine DNA (Ad/PEI/DNA) and mannose polyethylenimine DNA (ManPEI/DNA) complexes. Ad/PEI/DNA complexes have plasmid DNA bound to adenovirus particles by PEI and deliver DNA into cells via the adenovirus infection route. Such transfection complexes yield high transduction levels and sustained expression of luciferase and green fluorescent protein reporter genes and were almost as effective as recombinant adenovirus vectors. ManPEI/DNA complexes rely on uptake by receptor-mediated endocytosis via mannose receptor, which is highly expressed on DCs. While gene delivery by ManPEI/DNA complexes was less efficient than by Ad/PEI transfection, incorporation of adenovirus particles in ManPEI/DNA transfection complexes further enhanced transduction efficiencies and transgene expression. We also demonstrate that Ad/PEI-transfected DCs are competent in stimulating T cell proliferation in allogeneic and autologous mixed lymphocyte reactions, and in activating T cells from T cell receptor (TCR)-transgenic mice in an antigen-specific manner. Thus, the present study establishes the following relative order of transduction efficiencies of viral and nonviral gene delivery systems for primary human DCs: recombinant adenovirus > Ad/PEI = Ad/ManPEI > ManPEI > PEI. Ad/PEI and ManPEI transfection modes represent particularly versatile transduction systems for DCs, with ManPEI being built up exclusively of synthetic compounds.

Molecular cloning, expression and evolutionary analysis of the avian tyrosine kinase JAK1.

The Janus protein tyrosine kinases (JAK) constitute a protein family that plays a pivotal role in signalling of a large number of cytokine receptors. The cDNA of the chicken homologue of JAK1 was cloned and its nucleotide sequence determined. Chicken JAK1 protein comprises 1150 amino acids as deduced from its cDNA sequence with a calculated molecular mass of 133kDa. The overall structure of JAK proteins exemplified by the JAK homology domains JH1-JH7 is also preserved in chicken JAK1. Additionally, phylogenetic analysis demonstrates that chicken JAK1 is more closely related to mammalian JAK1 than to those of fish, exhibiting 80%, 79% and 63% identity in amino acid sequence to human, mouse and zebrafish JAK1, respectively. JAK1 proteins were found to be most conserved in the kinase (JH1) and pseudokinase (JH2) domains. This data is supported by Southern hybridization studies of ZOO blots. Chicken JAK1 shows a ubiquitous expression pattern and is transcribed as a 5.5kb mRNA in various tissues and cell types. JAK1 expression was particularly high in lymphoid cells.

Molecular cloning, expression and regulation of the avian tubby-like protein 1 (tulp1) gene.

The tubby-like protein 1 (tulp1) gene is a member of the tubby multigene family which includes tub, tulp1, tulp2 and tulp3. Human and mouse tulp1 genes were cloned and mutations in tulp1 have been implicated in retinitis pigmentosa in man. Here we report on the cDNA cloning of the chicken tulp1 homologue and its protein product deduced from the nucleotide sequence. The chicken Tulp1 protein comprises 358 amino acids with a calculated molecular mass of 40 kDa. The overall structure of Tub and Tulp proteins, exemplified by the highly conserved C-terminal domain of 255 amino acids and the signature motif KLACE, is also preserved in chicken Tulp1. Phylogenetic analysis demonstrates that chicken tulp1 cDNA and protein are closely related to human and mouse tulp1. In addition, chicken tulp1 mRNA is abundantly expressed in retina similar to tulp1 expression in human and mouse. Two tulp1-specific transcripts of 2 and 3 kb in size were identified that showed differential regulation during embryonic and postnatal development. Finally, tulp1 mRNA was found to be expressed in chicken erythroid cells and upregulated by ligand-activated thyroid hormone receptor (TR alpha/c-erbA).

Dendritic cells conditionally transformed by v-relER oncogene express lymphoid marker genes.

The initiation of primary immune responses is the key function of specialized antigen presenting cells, the dendritic cells (DC). DC of myeloid origin capture antigens in tissues, migrate to lymphoid organs and stimulate T cell responses. A subset of DC has been described which expresses lymphoid determinants and has potential regulatory functions. Conditional transformation of chicken bone marrow progenitors with v-relER, a v-rel estrogen receptor (ER) fusion gene, allows expansion of progenitors that can be induced to differentiate into DC in vitro. In this paper we describe that v-relER cells exhibit both myeloid and lymphoid surface markers, while B cell, T cell and NK (natural killer)-specific surface markers are absent. v-relER DC express, however, cytoplasmic CD3 protein and mRNA for CD8alpha and the lymphoid transcription factor GATA-3. These data suggest that v-relER DC might be related to the lymphoid subset of DC described in mammals.

[A case of cerebral polyuric hyponatremia (author's transl)].

A case of cerebral polyuric hyponatremia was reported. A 64-year-old lady had complained of visual disturbance for 4 years. She was operated upon under the diagnosis of tuberculum sellae meningioma. Sever days following operation tremendous polyuria was senn, i. e. polyuria over 40 l/day, massive sodium wasting in the urine, hyponatremia and severe thirst feeling. These situations could not easily controlled by pitressin, and 2 days after the onset of polyuria the level of sodium in the serum decreased up to 112 mEq/l, the amount of excretion of sodium into urine increased up to 186 mEq/l, and the patient had finally a convulsive seizure followed by loss of consciousness. This hyponatremia was easily controlled by infusion of hypertonic NaCl. The pathogenesis of this condition could not be explained by applying the classical concept of "Diabetes Insipidus" nor "SIADH". This hyponatremia might be the result of massive sodium wasting in the urine. And from the results obtained in our patient, these conditions should be classified in the syndrome of "Cerebral Polyuric Hyponatremia" as reported by Oi et al. It should be emphasized that the measurement of osmorality and level of electrolytes in the serum and urine had to be performed frequently when the postoperative polyuric situation was found, so that the clinical diagnosis could be established in the earlier stage. The clinical diagnosis and differentiation of postoperative disorders in water and electrolytes of central origin were discussed.

Epimutations mimic genomic mutations of DNMT3A in acute myeloid leukemia.

Mutations in the genetic sequence of the DNA de novo methyltransferase DNMT3A (DNA methyltransferase 3A) are found in many patients with acute myeloid leukemia (AML). They lead to dysfunction of DNMT3A protein and represent a marker for poor prognosis. Effects of genetic mutations can be mimicked by epigenetic modifications in the DNA methylation (DNAm) pattern. Using DNAm profiles of the Cancer Genome Atlas Research Network (TCGA), we identified aberrant hypermethylation at an internal promoter region of DNMT3A, which occurred in about 40% of AML patients. Bisulfite pyrosequencing assays designed for this genomic region validated hypermethylation specifically in a subset of our AML samples. High DNAm levels at this site are particularly observed in samples without genetic mutations in DNMT3A. Epimutations and mutations of DNMT3A were associated with related gene expression changes such as upregulation of the homeobox genes in HOXA and HOXB clusters. Furthermore, epimutations in DNMT3A were enriched in patients with poor or intermediate cytogenetic risk, and in patients with shorter event-free survival and overall survival (OS). Taken together, aberrant DNA hypermethylation within the DNMT3A gene, in analogy to DNMT3A mutations, is frequently observed in AML and both modifications seem to be useful for risk stratification or choice of therapeutic regimen.

A responsive MRI contrast agent to monitor functional cell status.

It has been shown that insoluble Gd chelates are suitable MRI contrast agents for conditional activation by intracellular lipases. The DTPA-based, insoluble, inactive contrast agent was internalized into dendritic cells by phagocytosis. Cleavage of long aliphatic side chains by intracellular lipase activity leads to the contrast agents solubility and hereby its activation depending on the enzyme expression. Uptake and activation of the contrast agent was much reduced in Flt3+ CD11b+ progenitor cells. Detectability limits in the T(1)-weighted MR images were estimated in phantoms and in vivo in the rat brain. Marginal toxic effects were only observed at very high concentrations of the contrast agent. The chelate can easily be modified to be targeted by enzymes expressed during specific change of cell status like activation or differentiation. Such a system is suitable for functional cellular in vivo MR imaging.