Christoph Hansis, M.D., Ph.D.

Derivation of novel genetically diverse human embryonic stem cell lines
Hansis C.; Lehmann R.
2011 ;96(3 SUPPL 1):S87-S87, Fertility & sterility
OBJECTIVE: Since many of the human embryonic stem cell (hESC) lines derived to date are poorly characterized, unavailable or do not represent desired traits, we generated twelve novel genetically diverse hESC lines with IRB approval and performed in-depth characterization of their molecular and cellular features. DESIGN: Experimental MATERIALS AND METHODS: Zona-free human blastocysts or inner cell masses (ICMs) (n = 60), some of them previously assessed by preim- plantation genetic diagnosis (PGD) for genetic conditions, were plated onto feeder cells and cultured in DMEM-based media. Differentiation of hESCs was achieved by colony overgrowth or embryoid body formation. Embryonic and control cells were subjected to marker gene, protein and chromosome analysis for pluripotency, differentiation, DNA fingerprinting and mutation examination by reverse transcription and genomic PCR, immu- nofluorescence, fluorescence in situ hybridization (FISH) and microarray analysis, respectively. RESULTS: Colonies (n = 21; 35% of plated blastocysts/ICMs) andhESC lines (n = 12; 57% of colonies) could be established, seven of which carrying disease-specific mutations (one each of trisomy 14, trisomy 17, trisomy X, 18, 21, 22, alpha-thalassemia X-linked mental retardation syndrome, Zell- weger syndrome and two of unbalanced translocation long arm of chromosomes 8, 15) and one derived from an inconclusively tested embryo (BRCA1). HESC lines harbored diverse ethnic heritage as well as linkage to conditions with a genetic component such as asthma or poor sperm morphology. Genetic abnormalities, gender, X chromosome activation status and DNA fingerprint and growth pattern could be assessed. Furthermore, hESCs could be differentiated into a variety of cell types, including the tissues most affected by the conditions. CONCLUSION: Our detailed analyses showed that we could establish novel genetically diverse hESC lines. Those lines will provide new tools for basic research, disease modeling, regenerative medicine, drug discovery and toxicity testing
— id: 150887, year: 2011, vol: 96, page: S87, stat: Journal Article,

Derivation of Novel Genetically Diverse Human Embryonic Stem Cell Lines
Stefanova VT; Grifo JA; Hansis C
2011 Dec 28;:?-? #, Stem cells & development
Human embryonic stem cells (hESCs) have the potential to revolutionize many biomedical fields ranging from basic research to disease modeling, regenerative medicine, drug discovery and toxicity testing. A multitude of hESC lines have been derived worldwide since the first five lines by Thomson and colleagues 13 years ago, but many of these are poorly characterized, unavailable or do not represent desired traits, thus making them unsuitable for application purposes. In order to provide the scientific community with better options, we have derived twelve new hESC lines at New York University from discard genetically normal and abnormal embryos using the latest techniques. We examined the genetic status of the NYUES lines in detail as well as their molecular and cellular features and DNA fingerprinting profile. Furthermore, we differentiated our hESCs into the tissues most affected by a specific condition or into clinically desired cell types. To our knowledge, a number of characteristics of our hESCs have not previously been reported, e.g. mutation for alpha thalassemia X-linked mental retardation syndrome, linkage to conditions with a genetic component such as asthma or poor sperm morphology and novel combinations of ethnic backgrounds. Importantly, all of our undifferentiated euploid female lines tested to date did not show X chromosome inactivation, believed to result in superior potency. We continue to derive new hESC lines and add them to the NIH registry and other registries. This should facilitate the use of our hESCs and lead to advancements for patient-benefitting applications
— id: 149781, year: 2011, vol: , page: ?, stat: Journal Article,

GENERATION AND CHARACTERIZATION OF DISEASE-SPECIFIC HUMAN EMBRYONIC STEM CELLS FROM GENETICALLY ABNORMAL EMBRYOS
Hansis, C.; Rice, C. E.; Lehmann, R.; Grifo, J. A.
2010 SEP ;94(4):S30-S30, Fertility & sterility
— id: 113763, year: 2010, vol: 94, page: S30, stat: Journal Article,

Novel highly efficient generation of disease-specific human embryonic stem cells from genetically abnormal embryos
Hansis C.; Rice C.E.; Grifo J.A.; Lehmann R.
2009 ;92(3 SUPPL 1):S86-S86, Fertility & sterility
OBJECTIVE: Since many frequent, fatal, and incurable diseases do not have an appropriate disease model and with attainable embryos being scarce, with IRB approval we attempted to generate novel disease-specific human embryonic stem (hES) cells with new protocols for more efficient derivation, maintenance, and differentiation. DESIGN: Experimental. MATERIALS AND METHODS: Zona-free human blastocysts (n=14) previously assessed by preimplantation genetic diagnosis (PGD) for genetic conditions were transferred onto feeder cells and cultured in DMEM-based media. Pieces of the colonies (n=4) were frozen by liquid nitrogen vitrification with cryoprotectants propylene glycol, DMSO, and acetamide and subsequently thawed. Differentiation of hES cells was achieved by colony overgrowth or embryoid body formation. Embryonic and control cells were subjected to marker gene and protein analysis for pluripotency and differentiation by reverse transcription PCR and immunofluorescence, respectively. RESULTS: Colonies (n=10; 71.4% of transferred blastocysts) could be established and maintained which showed the typical morphological features of hES cells such as compact colony formation. Colonies were derived from affected embryos (one each of cystic fibrosis, trisomy X, 18, 21, 22, and Tay-Sachs disease), from embryos tested inconclusively (one of cystic fibrosis and three of Tay-Sachs disease), and from three normal control embryos. Marker gene and protein expression as well as growth pattern analysis suggest that the colony cells retain their undifferentiated state in culture as well as after vitrification and thawing and that they can be differentiated into a variety of cell types, including the tissues most affected by the conditions. CONCLUSIONS: These newly established protocols for the derivation, maintenance, and differentiation of novel disease-specific hES cell lines should enable the efficient generation of new disease models. This will provide new tools to study diseases as well as to develop new therapeutic approaches
— id: 127243, year: 2009, vol: 92, page: S86, stat: Journal Article,

Totipotency, cell differentiation and reprogramming in humans
Hansis, Christoph
2006 Oct;13(4):551-557, Reproductive biomedicine online
Understanding the molecular mechanisms defining totipotency and cell differentiation in humans is a promising strategy in order to expand knowledge about reprogramming. Totipotency and the very first steps of cell differentiation can be studied well in early human embryos. Based on analysis of marker genes such as Oct-4 and -HCG, blastomeres seem to differ in their potency and can be regarded as lineage-specific stem cells as early as the 4-cell stage. The allocation of these stem cells to specific fates might hereby follow a pattern reminiscent of animal and vegetal poles. On the opposite end of the developmental spectrum, differentiated human cells can be used as a means of studying nuclear reprogramming. Intact human 293T kidney cells and primary leukocytes were reprogrammed towards a more undifferentiated state by Xenopus laevis egg extract. Molecular screens identified the chromatin-remodelling ATPase BRG1 as a factor required for this process. Based on these results, more efficient reprogramming protocols allowing for the generation of fully differentiated or undifferentiated human cells for clinical application may be developed
— id: 71566, year: 2006, vol: 13, page: 551, stat: Journal Article,

Initial differentiation of blastomeres in 4-cell human embryos and its significance for early embryogenesis and implantation
Edwards, Robert G; Hansis, Christoph
2005 Aug;11(2):206-218, Reproductive biomedicine online
This brief review is devoted to the nature of early blastomere differentiation in human 4-cell embryos and its consequences for embryonic development. Precursor cells of inner cell mass, germline, and trophectoderm may be formed at this stage, the clearest evidence being available for trophectoderm. The sites of these precursor cells in the embryo could be ascertained using markers for animal and vegetal poles, observing specific cleavage planes, and assessing gene and protein expression. This opens new opportunities for studying 4-cell embryos and removing or replacing specific cells. Knowledge of the properties of individual blastomeres should help in improving assisted human reproduction, performing preimplantation genetic diagnosis, and perhaps establishing specific stem cell lines. Special attention is paid to well-characterized trophectoderm, the trophectoderm stem cell, and possible new forms of clinical application
— id: 71561, year: 2005, vol: 11, page: 206, stat: Journal Article,

Nuclear reprogramming of human somatic cells by xenopus egg extract requires BRG1
Hansis, Christoph; Barreto, Guillermo; Maltry, Nicole; Niehrs, Christof
2004 Aug 24;14(16):1475-1480, Current biology. CB
Animal cloning by nuclear transplantation in amphibia was demonstrated almost half a century ago and raised the question of the mechanisms and genes involved in nuclear reprogramming. Here, we demonstrate nuclear reprogramming of permeabilized human cells using extracts from Xenopus laevis eggs and early embryos. We show upregulation of pluripotency markers Oct-4 and germ cell alkaline phosphatase (GCAP) in 293T cells and human primary leukocytes. Reprogrammed leukocytes had a limited life span and did not express surface antigens characteristic of pluripotent cells, indicating that reprogramming was incomplete. Reprogramming activity was detected in egg and early embryo extracts until early blastula stage. Late blastula-stage extracts were not only inactive but also inhibitory to reprogramming. Screening for factors required for reprogramming identified the chromatin remodeling ATPase BRG1. Antibody depletion of BRG1 protein or expression of dominant-negative BRG1 abolished the reprogramming ability of amphibian extracts. Conversely, overexpression of BRG1 in Xenopus animal caps extended their competence from blastula to gastrula stage to respond to basic fibroblast growth factor (bFGF) treatment with induction of the mesodermal marker Xbra. Dissection of the molecular machinery using a simplified assay system may aid in achieving complete nuclear reprogramming of somatic cells for regenerative medicine
— id: 71550, year: 2004, vol: 14, page: 1475, stat: Journal Article,

Candidate lineage marker genes in human preimplantation embryos
Hansis, Christoph; Grifo, James A; Krey, Lewis C
2004 May;8(5):577-583, Reproductive biomedicine online
Cell allocation and subsequent lineage commitment in the human embryo may be established as early as in the unfertilized oocyte. This phenomenon might be the result of subtle differences of gene expression and protein distribution. To assess whether gene expression profiling by reverse transcription-polymerase chain reaction could be a suitable tool for the detection of cell allocation and lineage commitment, the expression pattern of the putative inner cell mass marker gene Oct-4 and the trophectodermal marker genes beta-HCG and beta-LH were correlated in individual blastomeres of preimplantation human embryos. In 2- to 5-cell stage embryos, expression of beta-HCG and Oct-4 mRNA was negatively correlated in all blastomeres with statistical significance, suggesting that cell allocation can be assessed by those markers at early stages. In 7- to 10-cell stage embryos, expression of beta-LH and Oct-4 mRNA was negatively correlated in some blastomeres without statistical significance, suggesting that more experiments are necessary to decide if lineage commitment can be assessed in some cells by those markers at later stages
— id: 47784, year: 2004, vol: 8, page: 577, stat: Journal Article,

Cell differentiation in the preimplantation human embryo
Hansis, Christoph; Edwards, Robert G
2003 Mar;6(2):215-220, Reproductive biomedicine online
This brief paper analyses current knowledge on gene expression in individual blastomeres of preimplantation mammalian embryos. Initially, current knowledge on axes and cleavage planes in mammalian eggs and embryo blastomeres is described, together with gene and system homologies with flies and nematodes, and their influence on differentiation. Stress is placed on the need to study individual blastomeres, and even specific components within blastomeres. Examples of published work concentrate on the possible allocation of a single founder blastomere for trophectoderm, which contains large amounts of maternal leptin, STAT3 and other proteins positioned at the animal pole. The recent discovery that single human blastomeres in cleaving embryos contain high levels of HCGbeta mRNA and LHbeta mRNA suggests these are also trophectoderm foundation cells. It is now essential to discover if the maternal proteins leptin/STAT3 and maternal/embryonic HCGbeta transcripts locate to the same blastomere. Problems in jointly identifying maternal proteins and embryonic and maternal transcripts for specific proteins within one cell, and the nature of early cell allocation in mouse and human embryo, are discussed
— id: 71544, year: 2003, vol: 6, page: 215, stat: Journal Article,

SPEER--a new family of testis-specific genes from the mouse
Spiess, Andrej-Nikolai; Walther, Norbert; Muller, Nadine; Balvers, Marga; Hansis, Christoph; Ivell, Richard
2003 Jun;68(6):2044-2054, Biology of reproduction
Differential cloning revealed a partial mRNA sequence expressed in the mouse testis, which on further molecular characterization proved to be a member of a new family of 14 transcribed genes. Six of the genes appear to be expressed pseudogenes. The remainder indicate an open reading frame of approximately 200-220 amino acids encoding proteins with a very high proportion of alpha helical secondary structure, comprising approximately 15% glutamate residues. Because of this property, the family has been named SPErm-associated glutamate (E)-Rich protein (SPEER). Three members were chosen for more detailed characterization: SPEER-1 (pseudogene), SPEER-2, and SPEER-4D. All three are expressed tissue specifically in the testis of mice, with only very weak expression evident in the rat testis but in no other species tested. Using reverse transcription-polymerase chain reaction (RT-PCR), all three transcripts can be detected also in the epididymis, presumably due to the presence of spermatozoa. All three transcripts are expressed to high levels in haploid germ cells at the spermatocyte-spermatid transition. SPEER-1 mRNA is present in the cytoplasm as a sense transcript, SPEER-2 appears to be made mostly as an antisense transcript, whereas SPEER-4D appears to be localized within a subcellular compartment as a conventional sense transcript. Codon usage analysis suggests that all but the pseudogenes can be expressed as protein, confirmed for SPEER-2 and SPEER-4D by in vitro transcription/translation. An antibody raised against a peptide region of SPEER-4D, which probably cross-reacts with other SPEER members, immunohistochemically stains the nuclei of early round spermatids. While there are no true homologies to other proteins in the genome databases, some motifs are present that suggest a relationship to nuclear matrix proteins, implying that the SPEER family is a new group of haploid sperm-specific nuclear factors
— id: 71541, year: 2003, vol: 68, page: 2044, stat: Journal Article,

Assessment of beta-HCG, beta-LH mRNA and ploidy in individual human blastomeres
Hansis, Christoph; Grifo, James A; Tang, YaXu; Krey, Lewis C
2002 Sep-Oct;5(2):156-161, Reproductive biomedicine online
In human embryos, blastomeres differentiate into trophectoderm (TE) cells and inner cell mass (ICM) cells of blastocysts. Although morphologically indistinguishable, blastomeres at early cleavage stages are likely to undergo changes on a molecular level that make them destined to become ICM or TE cells. While the transcription factor Oct-4 might serve as a marker for totipotent ICM cells, human chorionic gonadotrophin might be used as the equivalent for TE cells. This study reports a reverse transcription-polymerase chain reaction procedure to assess human beta-HCG mRNA concentrations as well as ploidy in individual blastomeres from normally and abnormally fertilized human embryos. beta-HCG mRNA was detected in both euploid and aneuploid cells and in oocytes. Surprisingly, beta-LH mRNA was also detected in some euploid blastomeres. In regard to preimplantation genetic diagnosis, assessment of expression levels of beta-HCG and Oct-4 mRNA in individual biopsied cells might serve as a tool to identify embryogenic blastomeres in combination with testing for chromosome and single gene abnormalities
— id: 38846, year: 2002, vol: 5, page: 156, stat: Journal Article,

Analysis of Oct-4 expression and ploidy in individual human blastomeres
Hansis C; Tang YX; Grifo JA; Krey LC
2001 Feb;7(2):155-161, Molecular human reproduction
Oct-4, a decisive factor that maintains totipotency in murine embryonic and germ cells, is exclusively expressed in such cells. In mice, different levels of oct-4 expression in blastomeres predict development towards inner cell mass (ICM) (high oct-4) or trophectoderm (TE) (low oct-4). To address whether the mouse model also applies to human embryos, the cytoplasm of individual human blastomeres from normally and abnormally fertilized embryos was tested for Oct-4 expression by reverse transcription-polymerase chain reaction (RT-PCR). The nuclei of the same blastomeres were subjected to fluorescence in-situ hybridization (FISH) to determine ploidy. A significant difference in Oct-4 mRNA levels was revealed between blastomeres. The distribution of blastomeres with high Oct-4 levels varied according to the cleavage stage of the embryo: the more blastomeres, the lower the percentage with high Oct-4 levels. Aneuploid blastomeres did not exhibit lower Oct-4 mRNA levels than diploid ones. Thus, differential Oct-4 expression in individual human blastomeres appears to direct cells towards the ICM or TE lineages without regard to chromosomal status. Oct-4 might be used as a marker in preimplantation genetic diagnosis to identify embryogenic blastomeres
— id: 21255, year: 2001, vol: 7, page: 155, stat: Journal Article,

Tay-Sachs disease and preimplantation genetic diagnosis
Hansis, C; Grifo, J
2001 ;44:311-315, Advances in genetics
— id: 97656, year: 2001, vol: 44, page: 311, stat: Journal Article,

Oct-4 expression in inner cell mass and trophectoderm of human blastocysts
Hansis C; Grifo JA; Krey LC
2000 Nov;6(11):999-1004, Molecular human reproduction
The expression of the transcription factor Oct-4 is thought to be one of the decisive factors that maintain totipotency in embryonic and germ cells. In mice, oct-4 is exclusively expressed in germ cells and totipotent cells of the embryo. In humans, Oct-4 is expressed in germ cells, embryonic stem cells and whole embryos at various stages of development. However, there is limited information about the distribution of Oct-4 expression in human embryos. In an attempt to address this issue, the inner cell mass (ICM) and trophectoderm (TE) of 17 human blastocysts were separated and Oct-4 mRNA expression individually assessed by reverse transcription-polymerase chain reaction (RT-PCR). In discarded blastocysts that developed from two pronuclear zygotes, the mean Oct-4 expression was 31 times higher in totipotent ICM cells than in differentiated TE cells. This finding suggests that, in accordance with data from the mouse, Oct-4 is highly expressed in human ICM cells as opposed to TE cells; this in turn supports the hypothesis that Oct-4 plays a similar role to maintain totipotency in these two species
— id: 47803, year: 2000, vol: 6, page: 999, stat: Journal Article,

The gene for the Alzheimer-associated beta-amyloid-binding protein (ERAB) is differentially expressed in the testicular Leydig cells of the azoospermic by w/w(v) mouse
Hansis, C; Jahner, D; Spiess, A N; Boettcher, K; Ivell, R
1998 Nov 15;258(1):53-60, European journal of biochemistry
In order to discover possible new testicular paracrine factors involved in the establishment of spermatogenesis, a modified differential display reverse transcription, polymerase chain reaction (DDRT-PCR) procedure was used to detect gene transcripts preferentially expressed in the testes of the azoospermic w/w(v) mutant mouse. One of the differentially expressed gene products showed partial similarity to members of the short-chain alcohol dehydrogenase family of enzymes. This cDNA fragment was used to obtain the full-length mouse cDNA sequence, which initially showed moderate similarity to a 20beta-steroid dehydrogenase from lower organisms, and later shown to have >85% similarity to a novel endoplasmic-reticulum-associated-binding protein (ERAB) from the human brain, implicated in Alzheimer's disease. A recently cloned bovine sequence also of high similarity suggests that this molecule might also represent an isozyme of 3-hydroxyacyl-CoA dehydrogenase. Using the mouse cDNA as probe, northern hybridization showed enrichment of the transcript to the testicular Leydig cells, and showed a specific approximately 20-fold enrichment in the azoospermic mouse testis. The level of the testicular ERAB transcript does not seem to change through puberty, suggesting that a lack of germ cells alone is not responsible for the up-regulation in the w/w(v) testis. Using the three-dimensional coordinates of the published 20beta-hydroxysteroid dehydrogenase structure as template, it was additionally possible to construct a molecular model of the novel protein which showed it to have a very similar structure to this enzyme, including the substrate-binding domain
— id: 97657, year: 1998, vol: 258, page: 53, stat: Journal Article,

Differential display PCR cloning of W/Wv-mutant testis specific genes
Hansis, C; Jahner, D; Spiess, A N; Ivell, R
1997 ;424:147-149, Advances in experimental medicine & biology
— id: 97658, year: 1997, vol: 424, page: 147, stat: Journal Article,