Attenuating iPSC reprogramming stress with dominant-negative BET peptides.

Generation of induced pluripotent stem cells (iPSCs) is inefficient and stochastic. The underlying causes for these deficiencies are elusive. Here, we showed that the reprogramming factors (OCT4, SOX2, and KLF4, collectively OSK) elicit dramatic reprogramming stress even without the pro-oncogene MYC including massive transcriptional turbulence, massive and random deregulation of stress-response genes, cell cycle impairment, downregulation of mitotic genes, illegitimate reprogramming, and cytotoxicity. The conserved dominant-negative (DN) peptides of the three ubiquitous human bromodomain and extraterminal (BET) proteins enhanced iPSC reprogramming and mitigated all the reprogramming stresses mentioned above. The concept of reprogramming stress developed here affords an alternative avenue to understanding and improving iPSC reprogramming. These DN BET fragments target a similar set of the genes as the BET chemical inhibitors do, indicating a distinct approach to targeting BET proteins.

Non-linear relationship between basal serum albumin concentration and cardiac arrest in critically ill patients with end-stage renal disease: a cross-sectional study.

OBJECTIVES: The aim of our study was to investigate the association between serum albumin concentration and the risk of cardiac arrest in critically ill patients with end-stage renal disease in the intensive care unit (ICU). DESIGN: This was a secondary analysis. SETTING: The Phillip electronic-ICU collaborative database from 2014 to 2015. PARTICIPANTS: This study included 4990 critically ill patients diagnosed with end-stage renal disease. PRIMARY AND SECONDARY OUTCOME MEASURES: The exposure of interest was serum albumin concentration. The outcome variable was cardiac arrest. RESULTS: A non-linear relationship was observed between serum albumin concentration and risk of cardiac arrest, with an inflection point of 3.26 g/dL after adjusting for potential confounders. The effect sizes and the CIs on the left and right sides of the inflection point were 0.88 (0.65 to 1.19) and 0.32 (0.16 to 0.64), respectively. CONCLUSIONS: Within an albumin range of 3.26-5.6 g/dL, each 1 g/dL increase in serum levels is associated with a 68% decrease of the risk of cardiac arrest in critically ill patients with end-stage renal disease.

Reprogramming Human Fibroblasts to Induced Pluripotent Stem Cells Using the GFP-Marked Lentiviral Vectors in the Chemically Defined Medium.

Much investigation is needed to understand the underlying molecular mechanisms of iPSC reprogramming and to improve this technology. Lentivirus-mediated iPSC reprogramming remains the most effective method to study human pluripotency reprogramming. iPSC production is more efficient and consistent in the chemically defined medium. Fibroblasts are the most common starting cells for iPSC generation. Here, we provide a detailed protocol for iPSC generation from human fibroblasts using the GFP-expressing lentiviral vectors in the chemically defined medium.

Evaluating Reprogramming Efficiency and Pluripotency of the Established Human iPSCS by Pluripotency Markers.

The pluripotency of human induced pluripotent stem cells (HiPSCs) cannot be tested strictly in a similar way as we can do for the mouse ones because of ethical restrictions. One common and initial approach to prove the pluripotency of an established human iPSC line is to demonstrate expression of a set of established surface and intracellular pluripotency markers. This chapter provides procedures of immunocytochemistry of the established HiPSC lines for a set of the signature intracellular pluripotency proteins, OCT4, SOX2, NANOG, and LIN28. We also describe cell phenotyping by flow cytometry for the five established human pluripotency surface markers, SSEA3, SSEA4, TRA-1-60, TRA-1-81, and TRA2-49 (ALP). Numbers of ALP+ and TRA-1-60+ colonies are the most widely used parameters for evaluation of human iPSC reprogramming efficiency. Therefore, this chapter also provides detailed steps for substrate colorimetric reaction of the ALP activity, as well as the TRA-1-60 staining, of the iPSC colonies in the reprogramming population.

Become Competent in Generating RNA-Seq Heat Maps in One Day for Novices Without Prior R Experience.

Heat map visualization of RNA-seq data is a commonplace task. However, most laboratories rely on bioinformaticians who are not always available. Biological scientists are afraid to prepare heat maps independently because R is a programming platform. Here, using RNA-seq data for 16 differentially expressed genes in WNT pathway between embryonic stem cells and fibroblasts, I share a tutorial for novices without any prior R experience to master the skills, in one day, required for preparation of heat maps using the pheatmap package. Procedures described include installation of R, RStudio, and the pheatmap package, as well as hands-on practices for some basic R commands, conversion of RNA-seq data frame to a numeric matrix suitable for generation of heat maps, and defining arguments for the pheatmap function to make a desired heat map. More than 20 template scripts are provided to generate heat maps and to control the dimensions and appearances of the heat maps.

Quick, Coordinated and Authentic Reprogramming of Ribosome Biogenesis during iPSC Reprogramming.

Induction of pluripotent stem cells (iPSC) by OCT4 (octamer-binding transcription factor 4), SOX2 (SR box 2), KLF4 (Krüppel-Like Factor 4), and MYC (cellular Myelocytomatosis, c-MYC or MYC) (collectively OSKM) is revolutionary, but very inefficient, slow, and stochastic. It is unknown as to what underlies the potency aspect of the multi-step, multi-pathway, and inefficient iPSC reprogramming. Mesenchymal-to-epithelial (MET) transition is known as the earliest pathway reprogrammed. Using the recently established concepts of reprogramome and reprogramming legitimacy, the author first demonstrated that ribosome biogenesis (RB) is globally enriched in terms of human embryonic stem cells in comparison with fibroblasts, the popular starting cells of pluripotency reprogramming. It is then shown that the RB network was reprogrammed quickly in a coordinated fashion. Human iPSCs also demonstrated a more robust ribosome biogenesis. The quick and global reprogramming of ribosome biogenesis was also observed in an independent fibroblast line from a different donor. This study additionally demonstrated that MET did not initiate substantially at the time of proper RB reprogramming. This quick, coordinated and authentic RB reprogramming to the more robust pluripotent state by the OSKM reprogramming factors dramatically contrasts the overall low efficiency and long latency of iPSC reprogramming, and aligns well with the potency aspect of the inefficient OSKM reprogramming.

Human transcription factors responsive to initial reprogramming predominantly undergo legitimate reprogramming during fibroblast conversion to iPSCs.

The four transcription factors OCT4, SOX2, KLF4, and MYC (OSKM) together can convert human fibroblasts to induced pluripotent stem cells (iPSCs). It is, however, perplexing that they can do so only for a rare population of the starting cells with a long latency. Transcription factors (TFs) define identities of both the starting fibroblasts and the end product, iPSCs, and are also of paramount importance for the reprogramming process. It is critical to upregulate or activate the iPSC-enriched TFs while downregulate or silence the fibroblast-enriched TFs. This report explores the initial TF responses to OSKM as the molecular underpinnings for both the potency aspects and the limitation sides of the OSKM reprogramming. The authors first defined the TF reprogramome, i.e., the full complement of TFs to be reprogrammed. Most TFs were resistant to OSKM reprogramming at the initial stages, an observation consistent with the inefficiency and long latency of iPSC reprogramming. Surprisingly, the current analyses also revealed that most of the TFs (at least 83 genes) that did respond to OSKM induction underwent legitimate reprogramming. The initial legitimate transcriptional responses of TFs to OSKM reprogramming were also observed in the reprogramming fibroblasts from a different individual. Such early biased legitimate reprogramming of the responsive TFs aligns well with the robustness aspect of the otherwise inefficient and stochastic OSKM reprogramming.

Become Competent within One Day in Generating Boxplots and Violin Plots for a Novice without Prior R Experience.

The boxplot is a powerful visualization tool of sampled continuous data sets because of its rich information delivered, compact size, and effective visual expression. The advantage of boxplots is not widely appreciated. Many top journals suggest that boxplots should be used in place of bar charts, but have been wrongly replaced by bar charts. One technical barrier to the usage of boxplots in reporting quantitative data is that bench scientists are not competent in generating boxplots, and are afraid of R, a programming tool. This tutorial provides an effective training material in that even a novice without prior R experience can become competent, within one day, in generating professional boxplots. The available R scripts for boxplots are very limited in scope and are aimed at specialists, and the bench scientists have difficulty in following these scripts. This tutorial provides extensive step-by-step R scripts and instructions, as well as 29 illustrations for customizing every detail of the boxplot structures. Basic R commands and concepts are introduced for users without prior R experiences, which can be skipped by audiences with R knowledge. Violin plots are the enhanced version of boxplots, and therefore, this tutorial also provides a brief introduction and usage of the R package vioplot with one additional illustration. While the protocol is prepared for the newbies and trainees it will be a handy tool for infrequent users, and may benefit the experienced users as well since it provides scripts for customizing every detail of boxplots.

Profiling and quantification of pluripotency reprogramming reveal that WNT pathways and cell morphology have to be reprogramed extensively.

Pluripotent state can be established via reprogramming of somatic nuclei by factors within an oocyte or by ectopic expression of a few transgenes. Considered as being extensive and intensive, the full complement of genes to be reprogrammed, however, has never been defined, nor has the degree of reprogramming been determined quantitatively. Here, we propose a new concept of reprogramome, which is defined as the full complement of genes to be reprogrammed to the expression levels found in pluripotent stem cells (PSCs). This concept in combination with RNA-seq enables us to precisely profile reprogramome and sub-reprogramomes, and study the reprogramming process with the help of other available tools such as GO analyses. With reprogramming of human fibroblasts into PSCs as an example, we have defined the full complement of the human fibroblast-to-PSC reprogramome. Furthermore, our analyses of the reprogramome revealed that WNT pathways and genes with roles in cellular morphogenesis should be extensively and intensely reprogrammed for the establishment of pluripotency. We further developed a new mathematical model to quantitate the overall reprogramming, as well as reprogramming in a specific cellular feature such as WNT signaling pathways and genes regulating cellular morphogenesis. We anticipate that our concept and mathematical model may be applied to study and quantitate other reprogramming (pluripotency reprogramming from other somatic cells, and lineage reprogramming), as well as transcriptional and epigenetic differences between any two types of cells including cancer cells and their normal counterparts.

A PIANO (Proper, Insufficient, Aberrant, and NO Reprogramming) Response to the Yamanaka Factors in the Initial Stages of Human iPSC Reprogramming.

Yamanaka reprogramming is revolutionary but inefficient, slow, and stochastic. The underlying molecular events for these mixed outcomes of induction of pluripotent stem cells (iPSC) reprogramming is still unclear. Previous studies about transcriptional responses to reprogramming overlooked human reprogramming and are compromised by the fact that only a rare population proceeds towards pluripotency, and a significant amount of the collected transcriptional data may not represent the positive reprogramming. We recently developed a concept of reprogramome, which allows one to study the early transcriptional responses to the Yamanaka factors in the perspective of reprogramming legitimacy of a gene response to reprogramming. Using RNA-seq, this study scored 579 genes successfully reprogrammed within 48 h, indicating the potency of the reprogramming factors. This report also tallied 438 genes reprogrammed significantly but insufficiently up to 72 h, indicating a positive drive with some inadequacy of the Yamanaka factors. In addition, 953 member genes within the reprogramome were transcriptionally irresponsive to reprogramming, showing the inability of the reprogramming factors to directly act on these genes. Furthermore, there were 305 genes undergoing six types of aberrant reprogramming: over, wrong, and unwanted upreprogramming or downreprogramming, revealing significant negative impacts of the Yamanaka factors. The mixed findings about the initial transcriptional responses to the reprogramming factors shed new insights into the robustness as well as limitations of the Yamanaka factors.

On Mammalian Totipotency: What Is the Molecular Underpinning for the Totipotency of Zygote?

The mammalian zygote is described as a totipotent cell in the literature, but this characterization is elusive ignoring the molecular underpinnings. Totipotency can connote genetic totipotency, epigenetic totipotency, or the reprogramming capacity of a cell to epigenetic totipotency. Here, the implications of these concepts are discussed in the context of the properties of the zygote. Although genetically totipotent as any diploid somatic cell is, a zygote seems not totipotent transcriptionally, epigenetically, or functionally. Yet, a zygote may retain most of the key factors from its parental oocyte to reprogram an implanted differentiated genome or the zygote genome toward totipotency. This totipotent reprogramming process may extend to blastomeres in the two-cell-stage embryo. Thus, a revised alternative model of mammalian cellular totipotency is proposed, in which an epigenetically totipotent cell exists after the major embryonic genome activation and before the separation of the first two embryonic lineages.

Reprogramming by De-bookmarking the Somatic Transcriptional Program through Targeting of BET Bromodomains.

One critical event in reprogramming to pluripotency is erasure of the somatic transcriptional program of starting cells. Here, we present the proof of principle of a strategy for reprogramming to pluripotency facilitated by small molecules that interfere with the somatic transcriptional memory. We show that mild chemical targeting of the acetyllysine-binding pockets of the BET bromodomains, the transcriptional bookmarking domains, robustly enhances reprogramming. Furthermore, we show that chemical targeting of the transcriptional bookmarking BET bromodomains downregulates or turns off the expression of somatic genes in both naive and reprogramming fibroblasts. Chemical blocking of the BET bromodomains also results in loss of fibroblast morphology early in reprogramming. We therefore experimentally demonstrate that cell fate conversion can be achieved by chemically targeting the transcriptional bookmarking BET bromodomains responsible for transcriptional memory.

The acetyllysine reader BRD3R promotes human nuclear reprogramming and regulates mitosis.

It is well known that both recipient cells and donor nuclei demonstrate a mitotic advantage as observed in the traditional reprogramming with somatic cell nuclear transfer (SCNT). However, it is not known whether a specific mitotic factor plays a critical role in reprogramming. Here we identify an isoform of human bromodomain-containing 3 (BRD3), BRD3R (BRD3 with Reprogramming activity), as a reprogramming factor. BRD3R positively regulates mitosis during reprogramming, upregulates a large set of mitotic genes at early stages of reprogramming, and associates with mitotic chromatin. Interestingly, a set of the mitotic genes upregulated by BRD3R constitutes a pluripotent molecular signature. The two BRD3 isoforms display differential binding to acetylated histones. Our results suggest a molecular interpretation for the mitotic advantage in reprogramming and show that mitosis may be a driving force of reprogramming.

The Universal 3D3 Antibody of Human PODXL Is Pluripotent Cytotoxic, and Identifies a Residual Population After Extended Differentiation of Pluripotent Stem Cells.

Podocalyxin-like protein (PODXL) is a member of CD34 family proteins. It is the protein that carries many post-translational epitopes responsible for various pluripotent surface markers including TRA-1-60, TRA-1-81, GCTM2, GP200, and mAb84. However, PODXL has not attracted the attention of stem cell biologists. Here, we report several features of PODXL mRNA and protein in pluripotent stem cells. Similar to the modification-dependent pluripotent epitopes, PODXL transcripts and carrier protein are also features of pluripotency. PODXL is highly expressed in early human embryos from oocytes up to four-cell stages. During reprogramming of human cells to pluripotency, in contrast to TRA-1-60 and TRA-1-81, PODXL is activated by KLF4 at a very early time of reprogramming. Although TRA-1-60 and TRA-1-81 are completely lost upon differentiation, a residual PODXL(+) population exists even after extended differentiation and they were identified by the universal human PODXL epitope 3D3. Unlike TRA-1-60 and TRA-1-81 epitopes that are unique to primate pluripotent stem cells (PSCs), PODXL carrier protein can be used as a murine surface marker. Most importantly, antibody to 3D3 epitope causes massive necrosis and apoptosis of human PSCs (hPSCs). We suggest that 3D3 antibody could be employed to eliminate the tumorigenic pluripotent cells in hPSC-derived cells for cell transplantation.

Ubiquitin regulates TORC1 in yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae the TOR complex 1 (TORC1) controls many growth-related cellular processes and is essential for cell growth and proliferation. Macrolide antibiotic rapamycin, in complex with a cytosol protein named FKBP12, specifically inhibits TORC1, causing growth arrest. The FKBP12-rapamycin complex interferes with TORC1 function by binding to the FRB domain of the TOR proteins. In an attempt to understand the role of the FRB domain in TOR function, we identified a single point mutation (Tor2(W2041R) ) in the FRB domain of Tor2 that renders yeast cells rapamycin resistant and temperature sensitive. At the permissive temperature, the Tor2 mutant protein is partially defective for binding with Kog1 and TORC1 is impaired for membrane association. At the restrictive temperature, Kog1 but not the Tor2 mutant protein, is rapidly degraded. Overexpression of ubiquitin stabilizes Kog1 and suppresses the growth defect associated with the tor2 mutant at the nonpremissive temperature. We find that ubiquitin binds non-covalently to Kog1, prevents Kog1 from degradation and stabilizes TORC1. Our data reveal a unique role for ubiquitin in regulation of TORC1 and suggest that Kog1 requires association with the Tor proteins for stabilization.

Discovery of survival factor for primitive chronic myeloid leukemia cells using induced pluripotent stem cells.

A definitive cure for chronic myeloid leukemia (CML) requires identifying novel therapeutic targets to eradicate leukemia stem cells (LSCs). However, the rarity of LSCs within the primitive hematopoietic cell compartment remains a major limiting factor for their study in humans. Here we show that primitive hematopoietic cells with typical LSC features, including adhesion defect, increased long-term survival and proliferation, and innate resistance to tyrosine kinase inhibitor (TKI) imatinib, can be generated de novo from reprogrammed primary CML cells. Using CML iPSC-derived primitive leukemia cells, we discovered olfactomedin 4 (OLFM4) as a novel factor that contributes to survival and growth of somatic lin(-)CD34(+) cells from bone marrow of patients with CML in chronic phase, but not primitive hematopoietic cells from normal bone marrow. Overall, this study shows the feasibility and advantages of using reprogramming technology to develop strategies for targeting primitive leukemia cells.

Vectorology and factor delivery in induced pluripotent stem cell reprogramming.

Induced pluripotent stem cell (iPSC) reprogramming requires sustained expression of multiple reprogramming factors for a limited period of time (10-30 days). Conventional iPSC reprogramming was achieved using lentiviral or simple retroviral vectors. Retroviral reprogramming has flaws of insertional mutagenesis, uncontrolled silencing, residual expression and re-activation of transgenes, and immunogenicity. To overcome these issues, various technologies were explored, including adenoviral vectors, protein transduction, RNA transfection, minicircle DNA, excisable PiggyBac (PB) transposon, Cre-lox excision system, negative-sense RNA replicon, positive-sense RNA replicon, Epstein-Barr virus-based episomal plasmids, and repeated transfections of plasmids. This review provides summaries of the main vectorologies and factor delivery systems used in current reprogramming protocols.

All roads lead to induced pluripotent stem cells: the technologies of iPSC generation.

Generation of induced pluripotent stem cells (iPSCs) via the ectopic expression of reprogramming factors is a simple, advanced, yet often perplexing technology due to low efficiency, slow kinetics, and the use of numerous distinct systems for factor delivery. Scientists have used almost all available approaches for the delivery of reprogramming factors. Even the well-established retroviral vectors confuse some scientists due to different tropisms in use. The canonical virus-based reprogramming poses many problems, including insertional mutagenesis, residual expression and re-activation of reprogramming factors, uncontrolled silencing of transgenes, apoptosis, cell senescence, and strong immunogenicity. To eliminate or alleviate these problems, scientists have tried various other approaches for factor delivery and transgene removal. These include transient transfection, nonintegrating viral vectors, Cre-loxP excision of transgenes, excisable transposon, protein transduction, RNA transfection, microRNA transfection, RNA virion, RNA replicon, nonintegrating replicating episomal plasmids, minicircles, polycistron, and preintegration of inducible reprogramming factors. These alternative approaches have their own limitations. Even iPSCs generated with RNA approaches should be screened for possible transgene insertions mediated by active endogenous retroviruses in the human genome. Even experienced researchers may encounter difficulty in selecting and using these different technologies. This survey presents overviews of iPSC technologies with the intention to provide a quick yet comprehensive reference for both new and experienced reprogrammers.

Generation of transgene-free iPSC lines from human normal and neoplastic blood cells using episomal vectors.

Human induced pluripotent stem cells (iPSCs) have become an important tool for modeling human diseases and are considered a potential source of therapeutic cells. Original methods for iPSC generation use fibroblasts as a cell source for reprogramming and retroviral vectors as a delivery method of the reprogramming factors. However, fibroblasts require extended time for expansion and viral delivery of transgenes results in the integration of vector sequences into the genome which is a source of potential insertion mutagenesis, residual expressions, and reactivation of transgenes during differentiation. Here, we provide a detailed protocol for the efficient generation of transgene-free iPSC lines from human bone marrow and cord blood cells with a single transfection of non-integrating episomal plasmids. This method uses mononuclear bone marrow and cord blood cells, and makes it possible to generate transgene-free iPSCs 1-3 weeks faster than previous methods of reprogramming with fibroblasts. Additionally, we show that this approach can be used for efficient reprogramming of chronic myeloid leukemia cells.

Efficient generation of transgene-free induced pluripotent stem cells from normal and neoplastic bone marrow and cord blood mononuclear cells.

Reprogramming blood cells to induced pluripotent stem cells (iPSCs) provides a novel tool for modeling blood diseases in vitro. However, the well-known limitations of current reprogramming technologies include low efficiency, slow kinetics, and transgene integration and residual expression. In the present study, we have demonstrated that iPSCs free of transgene and vector sequences could be generated from human BM and CB mononuclear cells using non-integrating episomal vectors. The reprogramming described here is up to 100 times more efficient, occurs 1-3 weeks faster compared with the reprogramming of fibroblasts, and does not require isolation of progenitors or multiple rounds of transfection. Blood-derived iPSC lines lacked rearrangements of IGH and TCR, indicating that their origin is non-B- or non-T-lymphoid cells. When cocultured on OP9, blood-derived iPSCs could be differentiated back to the blood cells, albeit with lower efficiency compared to fibroblast-derived iPSCs. We also generated transgene-free iPSCs from the BM of a patient with chronic myeloid leukemia (CML). CML iPSCs showed a unique complex chromosomal translocation identified in marrow sample while displaying typical embryonic stem cell phenotype and pluripotent differentiation potential. This approach provides an opportunity to explore banked normal and diseased CB and BM samples without the limitations associated with virus-based methods.