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Characterization of iPS cells with abnormal karyotype. A) Control iPSC-K3 cells were generated from human foreskin fibroblasts and have a normal diploid male (46, XY) karyotype. B) A sub-line of iPSC-K3 cells, referred to as iPSC-K3aneuploid, have an abnormal near-tetraploid karyotype as well as rearrangements in chromosome 17 (red arrow). The karyogram presented here is representative of the cells analyzed. C) Left panel shows a phase contrast micrograph of iPSC-K3aneuploid cells in culture. Immunocytochemistry revealed the presence of OCT4 (middle panel) and DAPI was used to identify cell nuclei. Scale bar = 100 μm. D) FACS analysis reveals that iPSC-K3aneuploid cells do not express a marker of differentiated cells, CD13 (left panel); however, pluripotent markers SSEA-4 (middle panel) and Tra-1-81 (right panel) are expressed on the surface of 99% and 96% of the cells, respectively. Isotype controls for each antibody are indicated by the red line, antibodies by the blue line.
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Background
The characterization of induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) routinely includes analyses of chromosomal integrity. The belief is that pluripotent stem cells best suited to the generation of differentiated derivatives should display a euploid karyotype; although, this does not appear to have been formally...
Contexts in source publication
Context 1
... routine analyses of a subculture of iPSC-K3 cells we identified a cell line that was extensively aneu- ploid ( Figure 1 and Table 1). In this culture, the majority of the cells approached a tetraploid state, but included both gain and loss of discrete chromosomes as well as chromosomal rearrangements. ...Context 2
... given that all cells approached tetraploidy, we favor an al- ternative explanation in which the cells originated clonally from a single tetraploid event and subsequently diverged in culture. Figure 1 shows karyograms of the parental iPSC- K3 line and a single cell from the aneuploid iPSC-K3 (iPSC-K3 aneuploid ) derivative that contains the majority of the chromosomal abnormalities that were identified and Table 1 describes the frequency of each abnormality. In addition to the majority of the cells that displayed a shared karyotype, a single non-clonal cell was identified with the karyotype 66,XXY,+1,+2,-3,+4,+5,+9 + 12,-14,-15,-16, dup (17)(q11.2q25)ins(17)(q25;q25q11.2),-19,-19,-21,-21,-22. ...Context 3
... the abnormal karyotype, the iPSC-K3 aneuploid cells formed colonies that displayed a morphology that is characteristic of human pluripotent stem cells and im- munocytochemistry revealed that the majority of cells within the colonies expressed the pluripotency marker OCT4 ( Figure 1C). In addition, FACS analyses revealed that close to all of the cells in the culture expressed the pluripo- tent cell surface markers SSEA-4 (99%) and Tra-1-81 (96%), but not the fibroblast marker CD13 ( Figure 1D). ...Context 4
... the abnormal karyotype, the iPSC-K3 aneuploid cells formed colonies that displayed a morphology that is characteristic of human pluripotent stem cells and im- munocytochemistry revealed that the majority of cells within the colonies expressed the pluripotency marker OCT4 ( Figure 1C). In addition, FACS analyses revealed that close to all of the cells in the culture expressed the pluripo- tent cell surface markers SSEA-4 (99%) and Tra-1-81 (96%), but not the fibroblast marker CD13 ( Figure 1D). ...Similar publications
The demonstration that pluripotent stem cells could be generated by somatic cell reprogramming led to wonder if these so-called induced pluripotent stem (iPS) cells would extend our investigation capabilities in the cancer research field. The first iPS cells derived from cancer cells have now revealed the benefits and potential pitfalls of this new...
Citations
... IPSCs were grown in feeder-free conditions in six-well Nunclon ® surface plates (Nunc) coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) and maintained in mTeSR1 media (Stem Cell Technologies, Vancouver, BC, Canada) at 37 • C with 5% CO 2 . The K3 line has been previously characterized [15]. Cells were passaged every 4 days using a dispase solution (Thermo Fisher Scientific, Waltham, MA, USA) and were checked daily for differentiation. ...
The generation of gastrointestinal tissues from human pluripotent stem cells has provided unprecedented insight into the molecular mechanisms that drive the patterning of the primitive gut tube. Previous work has identified bone-morphogenetic-protein (BMP) signaling as an important mediator of mid/hindgut versus foregut and hindgut versus midgut cell fate choice. Inhibition of BMP signaling during gut tube morphogenesis inhibits the expression of the pan-intestinal transcription factor CDX2. Treatment of CDX2+ mid/hindgut cultures with BMP patterns them into hindgut, which gives rise to colonic organoids (HCOs). While the role for BMP signaling is clear, the molecular mechanisms through which BMP signaling patterns the mid/hindgut and colon remain unclear. BMPs bind to BMP receptors, activating a signaling cascade that results in the activation of SMADs, which function as transcription factors. We hypothesized that one of these factors, SMAD1, would be necessary for establishing the CDX2 domain and the colon domain. Unexpectedly, endoderm derived from SMAD1-deficient induced pluripotent stem cells was capable of inducing CDX2 in response to WNT and FGF signaling. In addition, CDX2+ gut tube cultures could activate posterior HOX genes in response to BMP. However, examination of HCOs following cytodifferentiation revealed that SMAD1-deficient HCOs ectopically expressed small-intestinal markers despite expressing posterior HOX genes. These results indicate that there is redundancy of SMADs during early hindgut patterning but that SMAD1 is required for the inhibition of small-intestinal gene expression in HCOs.
... Human-induced pluripotent stem cells IPSC72.3 (obtained from the Cincinnati Children's Hospital Pluripotent Stem Cell Facility) and K3 (obtained from Medical University of South Carolina Cell Models Core) were grown in feeder-free conditions in six-well Nunclon ® surface plates (Thermo fisher Scientific, Waltham, MA, USA) coated with Matrigel (BD Biosciences) and maintained in mTeSR1 media (Stem Cell Technologies, Vancouver, Canada) at 37 • C with 5% CO 2 . Both of these lines have been previously characterized [34,35]. Cells were passaged every 4 days using Dispase solution (Thermo-Fisher Scientific) and were checked daily for differentiation. ...
Human intestinal organoids (HIOs) generated from human pluripotent stem cells hold great promise for modeling human development and as a possible source of tissue for transplantation. HIOs generate all of the main epithelial and mesenchymal cell types found in the developing human intestine and mature into intestinal tissue with crypts and villi following transplantation into immunocompromised mice. However, incomplete in vitro patterning and the presence of contaminating neurons could hinder their use for regenerative medicine in humans. Based on studies in model organisms, we hypothesized that the treatment of HIOs with all trans retinoic acid (ATRA) would improve their in vitro growth and patterning. We found that ATRA not only improved the patterning of HIOs, ATRA also increased organoid forming efficiency, improved epithelial growth, enriched intestinal subepithelial myofibroblasts (ISEMFs) and reduced neuronal contamination in HIOs. Taken together, our studies demonstrate how the manipulation of a single developmental signaling pathway can be used to improve the survival, patterning and cellular composition of HIOs.
... This raises the question of the real-world risk associated with hPSC-derived clinical products. Significant genetic diversity already exists within the population, with copy number variations being reported across multiple different tissue types [110][111][112][113][114], and it has been reported that individuals can carry a significant mutational burden in cancerrelated genes without them becoming pathogenic [115,116]. Furthermore, many cancers have been reported to have highly specific cells of origin [117]. ...
Human pluripotent stem cells (hPSC) are known to acquire chromosomal abnormalities, which range from point mutations to large copy number changes, including full chromosome aneuploidy. These aberrations have a wide-ranging influence on the state of cells, in both the undifferentiated and differentiated state. Currently, very little is known on how these abnormalities will impact the clinical translation of hPSC, and particularly their potential to prime cells for oncogenic transformation. A further complication is that many of these abnormalities exist in a mosaic state in culture, which complicates their detection with conventional karyotyping methods. In this review we discuss current knowledge on how these aberrations influence the cell state and how this may impact the future of research and the cells’ clinical potential.
... Beyond using this technique to evaluate the genetic content in hPSC, a variety of other fields could benefit from the ability to parse out the genetic content of a complex population of cells. Heterogeneity continues to be an obstacle for the effective understanding of tumour makeup for example, and our understanding of heterogeneity in humans continues to be challenged as an increasing number of cell types in healthy individuals are shown to be mosaic and carry a variety of CNVs [18][19][20][21] . Here we analysed 56 single cells simultaneously and identified 13 cells with 50 unique aberrations not found in the bulk analysis of the original cell population. ...
Human pluripotent stem cells (hPSCs) have significant levels of low-grade genetic mosaicism, which commonly used techniques fail to detect in bulk DNA. These copy number variations remain a hurdle for the clinical translation of hPSC, as their effect in vivo ranges from unknown to dangerous, and the ability to detect them will be necessary as the field advances. As such there is need for techniques which can efficiently analyse genetic content in single cells with higher throughput and lower costs. We report here on the use of the Fluidigm C1 single cell WGA platform in combination with shallow whole genome sequencing to analyse the genetic content of single hPSCs. From a hPSC line carrying an isochromosome 20, 56 single cells were analysed and found to carry a total of 50 aberrations, across 23% of cells, which could not be detected by bulk analysis. Aberrations were predominantly segmental gains, with a fewer number of segmental losses and aneuploidies. Interestingly, 40% of the breakpoints seen here correspond to known DNA fragile sites. Our results therefore demonstrate the feasibility of single cell shallow sequencing of hPSC and further expand upon the biological importance and frequency of single cell mosaicism in hPSC.
... It is possible that changes in the chromosomal content are important for maintaining genetic diversity in the hepatocyte population, which facilitates the adaptive response of the liver to various lesions. Noto et al. [103] used a human iPSC line with pronounced aneuploidy: most cells were tetraploid (79 ~ 87 chromosomes), while the line included losses and gains of individual chromosomes and chromosomal rearrangements (the karyotype obtained by analyzing 19 cells was presented as the following: XXYY,-3 [19],-6 [18],-7 [5],-8 [18],-11 [16],+12 [19],-13 [3],-14 [4], -15 [19],-16 [12],-17 [5],dup(17)(q11.2q25)ins(17)(q25; q25q11.2) [19],-1 [12],-19 [19],-22 [11]). ...
... q25q11.2) [19],-1 [12],-19 [19],-22 [11]). Despite the karyotype, cells formed colonies with characteristic morphology and immunohistochemistry of human pluripotent stem cells, and the line itself demonstrated the ability to differentiate into hepatocyto-like cells [103]. These data confirm that there is considerable flexibility in the chromosome content that is compatible with differentiation into liver cells. ...
Human induced pluripotent stem cells (iPSCs) are a promising source of cells for regenerative medicine, study of the pathogenesis of various diseases, screening of pharmacological drugs, and other clinical and basic research. However, the maintenance of the genetic stability of the cells during reprogramming, long-term culture, and directed differentiation is necessary for the use of iPSCs. Large chromosomal aberrations affect the quality of iPSCs most adversely, so the review focuses on the analysis of chromosomal abnormalities, including the recurrent aneuploidy; the sources of its origin, the effect of reprogramming, and long-term culture on the accumulation of chromosome aberrations are discussed. Cases of spontaneous correction of the iPSCs karyotype and the possibility of induced correction of the large chromosomal abnormalities by removing or silencing the extra homologue are considered.
... One advantage of whole liver engineering approach is that the immune rejection issue can be potentially solved by engaging patient-specific liver cells such as those derived from induced pluripotent stem cells (iPSCs) in the process of recellularization 9,10 . Differentiation of iPSCs has been established for hepatocytes [11][12][13] and more recently for cholangiocytes [14][15][16] . We have shown that human iPSCs induced to differentiate on decellularized liver matrix differentiate into hepatocytes more efficiently than those on Matrigel indicating feasibility of this strategy 10 . ...
Engineered liver grafts for transplantation with sufficient hepatic function have been developed both in small and large animal models using the whole liver engineering approach. However, repopulation of the bile ducts in the whole liver scaffolds has not been addressed yet. In this study, we show the feasibility of repopulating the bile ducts in decellularized rat livers. Biliary epithelial cells were introduced into the bile ducts of the decellularized liver scaffolds with or without hepatocytes in the parenchymal space. The recellularized grafts were cultured under perfusion for up to 2 days and histological analysis revealed that the biliary epithelial cells formed duct-like structures, with the viable hepatocyte mass residing in the parenchymal space, in an arrangement highly comparable to the native tissue. The grafts were viable and functional as confirmed by both albumin and urea assay results and the gene expression analysis of biliary epithelial cells in recellularized liver grafts. This study provides the proof-of-concept results for rat liver grafts co-populated with parenchymal and biliary epithelial cells.
... G-banding karyotyping revealed that 6 of the 9 lines had normal diploid 46, XY karyotype with no detectable abnormalities, with the other 3 having abnormalities in chromosome 8 or 10 in some of the cells examined (Supplementary Fig. 1B). It is worth to note that for toxicological applications, even large chromosomal aberrations such as aneuploidy are permissive for iPSC-derived hepatocytes (Noto et al. 2014). Human pathogen screening indicated that all the lines are negative for bacteria, fungi, mycoplasma, and viruses (Supplementary Table 1). ...
Human induced pluripotent stem cells (iPSCs) provide a potentially unlimited source of differentiated cells from individuals with specific genetic backgrounds. Using self-replicative RNA reprogramming technology, we generated nine iPSC lines from endothelial progenitor cells (EPCs) derived from blood samples of three different ethnicities: Black or African American, Latino or Hispanic, and Non-Hispanic White. The resulting iPSC lines showed normal karyotype in large part, expressed pluripotency marker genes, and spontaneously differentiated in vitro into the three germ layers. These iPSC lines offer the potential to generate tissues with ethnic diversity, and thus afford a valuable tool for ethnic-related toxicological applications.
... The factors that modulate differentiation propensity are many and wide ranging, though the effects are not always clear, for example not all chromosomal abnormalities negatively impact or alter differentiation. Copy number variations are even thought to be permissive, and perhaps beneficial in neurons (Knouse et al., 2014) and hepatocytes (Duncan et al., 2010(Duncan et al., , 2012Noto et al., 2014) further muddying the definition of what is genetically 'normal' or acceptable for further use. Even though the driver mutations have been identified for many recurring abnormalities, those mechanisms do not always play a role in the altered differentiation patterns. ...
Background:
Human pluripotent stem cell (hPSC) lines are known to have a bias in their differentiation. This gives individual cell lines a propensity to preferentially differentiate towards one germ layer or cell type over others. Chromosomal aberrations, mitochondrial mutations, genetic diversity and epigenetic variance are the main drivers of this phenomenon, and can lead to a wide range of phenotypes.
Objective and rationale:
Our aim is to provide a comprehensive overview of the different factors which influence differentiation propensity. Specifically, we sought to highlight known genetic variances and their mechanisms, in addition to more general observations from larger abnormalities. Furthermore, we wanted to provide an up-to-date list of a growing number of predictive indicators which are able to identify differentiation propensity before the initiation of differentiation. As differentiation propensity can lead to difficulties in both research as well as clinical translation, our thorough overview could be a useful tool.
Search methods:
Combinations of the following key words were applied as search criteria in the PubMed database: embryonic stem cells, induced pluripotent stem cells, differentiation propensity (also: potential, efficiency, capacity, bias, variability), epigenetics, chromosomal abnormalities, genetic aberrations, X chromosome inactivation, mitochondrial function, mitochondrial metabolism, genetic diversity, reprogramming, predictive marker, residual stem cell, clinic. Only studies in English were included, ranging from 2000 to 2017, with a majority ranging from 2010 to 1017. Further manuscripts were added from cross-references.
Outcomes:
Differentiation propensity is affected by a wide variety of (epi)genetic factors. These factors clearly lead to a loss of differentiation capacity, preference towards certain cell types and oftentimes, phenotypes which begin to resemble cancer. Broad changes in (epi)genetics, such as aneuploidies or wide-ranging modifications to the epigenetic landscape tend to lead to extensive, less definite changes in differentiation capacity, whereas more specific abnormalities often have precise ramifications in which certain cell types become more preferential. Furthermore, there appears to be a greater, though often less considered, contribution to differentiation propensity by factors such as mitochondria and inherent genetic diversity. Varied differentiation capacity can also lead to potential consequences in the clinical translation of hPSC, including the occurrence of residual undifferentiated stem cells, and the transplantation of potentially transformed cells.
Wider implications:
As hPSC continue to advance towards the clinic, our understanding of them progresses as well. As a result, the challenges faced become more numerous, but also more clear. If the transition to the clinic is to be achieved with a minimum number of potential setbacks, thorough evaluation of the cells will be an absolute necessity. Altered differentiation propensity represents at least one such hurdle, for which researchers and eventually clinicians will need to find solutions. Already, steps are being taken to tackle the issue, though further research will be required to evaluate any long-term risks it poses.
... It is possible that changes in the chromosomal content are important for maintaining genetic diversity in the hepatocyte population, which facilitates the adaptive response of the liver to various lesions. Noto et al. [103] used a human iPSC line with pronounced aneuploidy: most cells were tetraploid (79 ~ 87 chromosomes), while the line included losses and gains of individual chromosomes and chromosomal rearrangements (the karyotype obtained by analyzing 19 cells was presented as the following: XXYY,-3 [19],-6 [18],-7 [5],-8 [18],-11 [16],+12 [19],-13 [3],-14 [4], -15 [19],-16 [12],-17 [5],dup(17)(q11.2q25)ins(17)(q25; q25q11.2) [19],-1 [12],-19 [19],-22 [11]). ...
... q25q11.2) [19],-1 [12],-19 [19],-22 [11]). Despite the karyotype, cells formed colonies with characteristic morphology and immunohistochemistry of human pluripotent stem cells, and the line itself demonstrated the ability to differentiate into hepatocyto-like cells [103]. These data confirm that there is considerable flexibility in the chromosome content that is compatible with differentiation into liver cells. ...
The method discovered by the Yamanaka group to convert terminally differentiated cells into induced pluripotent stem cells (iPSC) has sparked an explosion of research in this field in the last decade. Opportunities have emerged for the study of human diseases in which PSC scores obtained from patients are used as a powerful platform for studying the pathogenesis of diseases, especially those that previously could not be studied at the cellular level due to the material inaccessibility. However, despite significant progress in this area, the security problem remains largely unresolved, and the main debatable issue is the impact of reprogramming on the genome stability. Although the functional consequences of instability are not always significant, the presence of genetic aberrations in iPSC complicates their biomedical use. Preserving the stable genome of iPSC is a key factor in most areas of practical application of this technology, such as cell therapy, disease modeling and pharmacological studies. In this connection, data on the features of the mechanisms of maintaining the stability of the genome and the specificity of the mutational process in iPSC are of interest, and this review is devoted to the analysis of this issue. Since the large chromosomal aberrations are most compromising quality of iPSC, the emphasis is on the consideration of genetic integrity at the chromosomal and sub-chromosomal levels. Data are presented about the features of the cell cycle and the response to DNA damage in reprogrammed cells affecting chromosome segregation and the appearance of rearrangements, as well as information on the effects of replicative and oxidative stress on the stability of the iPSC genome.
... Although the genomic integrity of iPSCs has limited their clinical applications, numerous reprogramming strategies were developed to minimize the disturbance of the iPSC genome. While aneuploidy is commonly associated with cell transformation, iPSCs that harbor abnormal chromosomal content retain the capacity to generate HLCs with high efficiency [115]. ...
The conversion of somatic cells to hepatocytes has fundamentally re-shaped traditional concepts regarding the limited resources for hepatocyte therapy. With the various induced pluripotent stem cell (iPSC) generation routes, most somatic cells can be effectively directed to functional stem cells, and this strategy will supply enough pluripotent material to generate promising functional hepatocytes. However, the major challenges and potential applications of reprogrammed hepatocytes remain under investigation. In this review, we provide a summary of two effective routes including direct reprogramming and indirect reprogramming from somatic cells to hepatocytes and the general potential applications of the resulting hepatocytes. Through these approaches, we are striving toward the goal of achieving a robust, mature source of clinically relevant lineages.
