iPSC Reprogramming Research Articles

Extended passaging increases the efficiency of neural differentiation from induced pluripotent stem cells

BackgroundThe use of induced pluripotent stem cells (iPSCs) for the functional replacement of damaged neurons and in vitro disease modeling is of great clinical relevance. Unfortunately, the capacity of iPSC lines to differentiate into neurons is highly variable, prompting the need for a reliable means of assessing the differentiation capacity of newly derived iPSC cell lines. Extended passaging is emerging as a method of ensuring faithful reprogramming. We adapted an established and efficient embryonic stem cell (ESC) neural induction protocol to test whether iPSCs (1) have the competence to give rise to functional neurons with similar efficiency as ESCs and (2) whether the extent of neural differentiation could be altered or enhanced by increased passaging.ResultsOur gene expression and morphological analyses revealed that neural conversion was temporally delayed in iPSC lines and some iPSC lines did not properly form embryoid bodies during the first stage of differentiation. Notably, these deficits were corrected by continual passaging in an iPSC clone. iPSCs with greater than 20 passages (late-passage iPSCs) expressed higher expression levels of pluripotency markers and formed larger embryoid bodies than iPSCs with fewer than 10 passages (early-passage iPSCs). Moreover, late-passage iPSCs started to express neural marker genes sooner than early-passage iPSCs after the initiation of neural induction. Furthermore, late-passage iPSC-derived neurons exhibited notably greater excitability and larger voltage-gated currents than early-passage iPSC-derived neurons, although these cells were morphologically indistinguishable.ConclusionsThese findings strongly suggest that the efficiency neuronal conversion depends on the complete reprogramming of iPSCs via extensive passaging.

Human induced pluripotent cells resemble embryonic stem cells demonstrating enhanced levels of DNA repair and efficacy of nonhomologous end-joining

To maintain the integrity of the organism, embryonic stem cells (ESC) need to maintain their genomic integrity in response to DNA damage. DNA double strand breaks (DSBs) are one of the most lethal forms of DNA damage and can have disastrous consequences if not repaired correctly, leading to cell death, genomic instability and cancer. How human ESC (hESC) maintain genomic integrity in response to agents that cause DSBs is relatively unclear. Adult somatic cells can be induced to “dedifferentiate” into induced pluripotent stem cells (iPSC) and reprogram into cells of all three germ layers. Whether iPSC have reprogrammed the DNA damage response is a critical question in regenerative medicine. Here, we show that hESC demonstrate high levels of endogenous reactive oxygen species (ROS) which can contribute to DNA damage and may arise from high levels of metabolic activity. To potentially counter genomic instability caused by DNA damage, we find that hESC employ two strategies: First, these cells have enhanced levels of DNA repair proteins, including those involved in repair of DSBs, and they demonstrate elevated nonhomologous end-joining (NHEJ) activity and repair efficacy, one of the main pathways for repairing DSBs. Second, they are hypersensitive to DNA damaging agents, as evidenced by a high level of apoptosis upon irradiation. Importantly, iPSC, unlike the parent cells they are derived from, mimic hESC in their ROS levels, cell cycle profiles, repair protein expression and NHEJ repair efficacy, indicating reprogramming of the DNA repair pathways. Human iPSC however show a partial apoptotic response to irradiation, compared to hESC. We suggest that DNA damage responses may constitute important markers for the efficacy of iPSC reprogramming.

X inactivation: a histone protects from reprogramming by the frog

Genes on the inactive X chromosome (Xi) of female mammals are repressed in a remarkably stable manner and reactivation of transcription is generally not observed unless the cell is reprogrammed to an early embryonic type. In this issue of The EMBO Journal , Pasque et al (2011) use a reprogramming system in frog oocytes to study the stability of Xi chromatin in cells of different developmental stages and identify the histone variant macroH2A as a factor preventing transcriptional activation.

Harnessing the potential of induced pluripotent stem cells for regenerative medicine

The discovery of methods to convert somatic cells into induced pluripotent stem cells (iPSCs) through expression of a small combination of transcription factors has raised the possibility of producing custom-tailored cells for the study and treatment of numerous diseases. Indeed, iPSCs have already been derived from patients suffering from a large variety of disorders. Here we review recent progress that has been made in establishing iPSC-based disease models, discuss associated technical and biological challenges, and highlight possible solutions to overcome these barriers. We believe that a better understanding of the molecular basis of pluripotency, cellular reprogramming and lineage-specific differentiation of iPSCs is necessary for progress in regenerative medicine.

Abstract 488: Reprogramming cancer cells into terminal differentiation via induced pluripotency

Abstract Induced pluripotent stem cells (iPSCs) are defined as somatic cells that have been “reprogrammed” (using either the four transcriptional factors Oct4, Sox2, Klf4 and c-Myc or Oct4, Sox2, Nanog, and Lin28) to exhibit embryonic stem cell-like pluripotent differentiation properties. The generation of iPSCs was a great achievement for the field of cell transplantation and tissue engineering. More recently multiple cancer cell lines (e.g., leukemic cells, melanoma cells & gastric cancer cells) have been successfully reprogrammed to the iPSC-state, at least as defined by (1) expression of genes specific to undifferentiated embryonic stem cells; and (2) pluripotency as defined by embryoid body formation in vitro and early markers of commitment to various differentiation lineages. However, the question that remains unanswered is: can the cancer derived iPSCs complete maturation along a given lineage, be it the lineage from which it was derived (i.e., originally prematurely blocked) or an alternative lineage? If the former were demonstrated it would in essence answer the question of whether global changes in chromatin architecture or ‘epigenetics’ (which are believed to underlie the mechanism of nuclear iPSC reprogramming) can supersede the specific genetic changes (i.e., mutations) in DNA that resulted in the lineage specific differentiation block and subsequent tumorigenesis. To attempt to answer these questions we generated iPSC-cancer cells from sarcoma cell lines representing undifferentiated sarcomas, osteosarcoma cells, liposarcoma cells, and sarcomas of unknown lineage. While the original sarcoma cell lines could not be differentiated in vitro into mature connective tissue cells, their iPSC-counterparts readily differentiated into multiple mature connective tissue lineages. Furthermore, assessment of proliferation indicated that acquisition of the mature phenotype was concurrent with cessation of proliferation; and thus abrogation of tumorigenicity. We are currently (1) performing gene expression, miRNA, and global DNA promoter methylation profiling in order to assess the mechanism by which iPSC-based reprogramming of cancer cells over-rides the inherent genetic mutations in these cells and restores the capacity for terminal differentiation; and (2) extending our work to carcinomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 488. doi:10.1158/1538-7445.AM2011-488

MyoD Gene Suppression by Oct4 Is Required for Reprogramming in Myoblasts to Produce Induced Pluripotent Stem Cells

Expression of the four transcription factors, that is, Oct4, Sox2, cMyc, and Klf4 has been shown to generate induced pluripotent stem cells (iPSCs) from many types of specialized differentiated somatic cells. It remains unclear, however, whether fully committed skeletal muscle progenitor cells (myoblasts) have the potency to undergo reprogramming to develop iPSCs in line with previously reported cases. To test this, we have isolated genetically marked myoblasts derived from satellite cell of adult mouse muscles using the Cre-loxP system (Pax7-CreER:R26R and Myf5-Cre:R26R). On infection with retroviral vectors expressing the four factors, these myoblasts gave rise to myogenic lineage tracer lacZ-positive embryonic stem cell (ESC)-like colonies. These cells expressed ESC-specific genes and were competent to differentiate into all three germ layers and germ cells, indicating the successful generation of myoblast-derived iPSCs. Continuous expression of the MyoD gene, a master transcription factor for skeletal muscle specification, inhibited this reprogramming process in myoblasts. In contrast, reprogramming myoblasts isolated from mice lacking the MyoD gene led to an increase in reprogramming efficiency. Our data also indicated that Oct4 acts as a transcriptional suppressor of MyoD gene expression through its interaction with the upstream enhancer region. Taken together, these results indicate that suppression of MyoD gene expression by Oct4 is required for the initial reprogramming step in the development of iPSCs from myoblasts. This data suggests that the skeletal muscle system provides a well-defined differentiation model to further elaborate on the effects of iPSC reprogramming in somatic cells.

Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) offer immense potential for regenerative medicine and studies of disease and development. Somatic cell reprogramming involves epigenomic reconfiguration, conferring iPSCs with characteristics similar to embryonic stem (ES) cells. However, it remains unknown how complete the reestablishment of ES-cell-like DNA methylation patterns is throughout the genome. Here we report the first whole-genome profiles of DNA methylation at single-base resolution in five human iPSC lines, along with methylomes of ES cells, somatic cells, and differentiated iPSCs and ES cells. iPSCs show significant reprogramming variability, including somatic memory and aberrant reprogramming of DNA methylation. iPSCs share megabase-scale differentially methylated regions proximal to centromeres and telomeres that display incomplete reprogramming of non-CG methylation, and differences in CG methylation and histone modifications. Lastly, differentiation of iPSCs into trophoblast cells revealed that errors in reprogramming CG methylation are transmitted at a high frequency, providing an iPSC reprogramming signature that is maintained after differentiation.

A Murine ESC-like State Facilitates Transgenesis and Homologous Recombination in Human Pluripotent Stem Cells

Murine pluripotent stem cells can exist in two functionally distinct states, LIF-dependent embryonic stem cells (ESCs) and bFGF-dependent epiblast stem cells (EpiSCs). However, human pluripotent cells so far seemed to assume only an epiblast-like state. Here we demonstrate that human iPSC reprogramming in the presence of LIF yields human stem cells that display morphological, molecular, and functional properties of murine ESCs. We termed these hLR5 iPSCs because they require the expression of five ectopic reprogramming factors, Oct4, Sox2, Klf4, cMyc, and Nanog, to maintain this more naive state. The cells are "metastable" and upon ectopic factor withdrawal they revert to standard human iPSCs. Finally, we demonstrate that the hLR5 state facilitates gene targeting, and as such provides a powerful tool for the generation of recombinant human pluripotent stem cell lines.