Subvert the Paradigm - Reprogramming the Cell: Part 5 | First successful production of induced pluripotent stem cells from adult somatic cells

Image courtesy: hetemeel.com

Just a few months after the landmark studies performed by three independent biologists - Yamanaka, Maherali and Wernig, researchers are back with results which may end saga of human embryonic stem cell (hESC) controversy. Last week, Yamanaka et al. have published results in Science journal ¹ showing successful transformation of human dermal fibroblasts into induced pluripotent stem cells (iPS) by retroviral transduction of same four transcription factors - Oct3/4, Sox2, Klf4, and c-Myc which were earlier used in mouse models.² In an another study, Thomson J. and group published study in journal Cell³ showing OCT4, SOX2, NANOG, and LIN28 mediated conversion of human somatic cells into iPS. Both studies yielded iPS which are exhibiting strong genetic and functional similarity to hESC. This could lead to paradigm shift in current methods which requires use of hESC, mainly in drug development and potential to treat degenerative diseases like Alzheimer's and many others. However, team leaders expect a long run before this new principle would be available widely across the globe for further applications.

To note,

1. Role of c-Myc as a potential teratogen needs to be tested in human iPS, particularly in-vivo which is way far considering existing controversy surrounding hESC.

2. Recently^{4, 5}, Oct4 is identified as a dispensable gene rather critical one in inducing pluripotency in adult somatic cells. Since both of above studies involved Oct4 reactivation, further research would be of importance to define role of Oct4 more precisely.

Reference:

1, 3: See Wikipedia edits under induced pluripotent stem cell for original article links

2: Related post on generations of iPS | Reprogramming the Cell - Serial posts 1 to 4

4: Lenger, C. J. et al. Oct4 is dispensable for somatic stem cell self-renewal. Cell Stem Cell 1, 403–415 (2007)

5: Oct4 is dispensable for somatic stem cell self-renewal | Nature Reports Stem Cells 18-Oct-2007
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RNAi and now, RNAa - The bubble effect

RNAi mediated gene silencing is now considered a pivotal theory which is one of the most promising method of gene therapy in coming years. Synthetic short hairpin / short interfering (shRNA / siRNA) or naturally present micro RNA (miRNA) mediates translational silencing of particular gene(s) occurring in cytoplasm (co-driven by RNA-induced silencing complex - RISC). What if similar process is happening within nucleus by naturally occurring miRNA? Then it's called transcriptional repression, although the outcome would be the identical - inhibiting target protein production. Few studies showed such nuclear phenomenon in recent years. The bubble (DNA>RNA>Protein) bursts.

Now, think of the mirror side if one can generate the bubble. RNA mediated gene activation?!? logic and not the serendipity! Scientists are currently arguing a lot on RNAa effect, that is RNA mediated transcription activation and thereby, promoting protein synthesis. After consecutive failed several attempts to convince this theory since 2004, young scientists from UCSF laboratory, Long-Cheng Li, Rajvir Dahiya and R F Place have showed small dsRNA induced transcriptional activation in human cells with supporting study from separate team at UT SouthWestern in Dec-2006. Although they have yet to depict exact mechanism of such phenomenon, if at all it is present in reality. Skeptics who have pioneered in RNAi theory believe this could be the indirect inhibition of repressor genes or another miRNA who is naturally responsible for certain protein inhibition (i.e.: Tumor growth proteins). Interestingly, new research of RNAa is becoming more common among fresh graduates rather masters who demand solid evidence until RNAi terminology can be grounded off.

To bubble more !?!, co-relate this idea with following: the central dogma of molecular biology, dynamic dna, dna replication and holiday junction model, reprogramming.

To be continued.....

Courtesy: Nature magazine (Read full news article for detailed explanation and references)

News report by Erika Check: RNA interference: Hitting the on switch | Nature 2007:448;855-858 | doi:10.1038/448855a

Reference:
1. Long-Cheng Li, Robert F. Place, Rajvir Dahiya, et al. | Small dsRNAs induce transcriptional activation in human cells | PNAS 2006 (Nov 14):103;17337-17342
2. Janowski AB, Younger ST, Hardy DB, et al. | Activating gene expression in mammalian cells with promoter-targeted duplex RNAs | Nature Chemical Biology 2007:3;166-173 | doi:10.1038/nchembio860
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ENCODE - Code within the code

Pillars of Creation (Eagle nebula M16; image by NASA)

The code is getting more enigmatic as scientists have begun to unwind it, now raising more curiosity and challenges to understand the beautiful life! The National Human Genome Research Institute (NHGRI)'s project ENCODE, the Encyclopedia Of DNA Elements, started in September 2003 is a multi-institutional, multiphasic, collaborative project has just finished its pilot phase, first of planned three phases (now remaining, a technology development phase and a planned production phase) which aims towards identification of functional elements and organization of entire genome rather genes of particular interest and thereby, having clear insight of human genome and linked expression. ENCODE team is conducting series of experimental procedures to identify not only genes that code for proteins but also for non-protein coding genes, regulatory elements controlling transcription of genes; and elements that maintain the structure of chromosomes and mediate the dynamics of their replication.

In the pilot phase, researchers focused on 44 targets, which together cover 29,998 kilobases (kb) of the human genome, equivalent to about 1 percent of the human genome sequence, or about 30 million of 3 billion DNA base pairs. Results of pilot project are challenging the established fundamentals, particularly of non-protein coding genes and their overlapping on protein-coding loci, lack of evolutionary constraint (that is, the rejection of mutations at a particular location) of genes and dynamism in DNA replication and structure of its supporting proteins (i.e. histone) influencing transcription and gene expression. As ENCODE is advancing further, better understanding of genome and importantly its functional structure would eventually helpful to sidestep current obstacles of applied genetics. Below are few lines from the original article (see citation) that may make you dream to see glimpse of dynamics within the pillars of creation!

The protein-coding component of these genes makes up just a small fraction of the human genome - 1.5 percent to 2 percent. Evidence exists that other parts of the genome also have important functions.

The majority of DNA in the human genome is transcribed into functional molecules, called RNA, and that these transcripts extensively overlap one another. This broad pattern of transcription challenges the long-standing view that the human genome consists of a relatively small set of discrete genes, along with a vast amount of so-called junk DNA that is not biologically active.

Simple view of the genome as having a defined set of isolated loci transcribed independently does not seem to be accurate.

ENCODE effort found about half of functional elements in the human genome do not appear to have been obviously constrained during evolution, at least when examined by current methods used by computational biologists. This lack of evolutionary constraint may indicate that many species' genomes contain a pool of functional elements, including RNA transcripts, that provide no specific benefits in terms of survival or reproduction. As this pool turns over during evolutionary time, researchers speculate it may serve as a "warehouse for natural selection" by acting as a source of functional elements unique to each species and of elements that perform the similar functions among species despite having sequences that appear dissimilar.

Regulatory sequences are just as likely to be located downstream of a transcription start site on a DNA strand as upstream.

Chromatin accessibility and histone modification patterns are highly predictive of both the presence and activity of transcription start sites.

Critical questions are raised by the presence of a large amount of unannotated transcription with respect to how the corresponding sequences are organized in the genome - do these reflect longer transcripts that include known loci, do they link known loci, or are they completely separate from known loci?

Original article:

Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project
The ENCODE Project Consortium
Nature 2007;447:799-816 (14 June 2007)
doi:10.1038/nature05874

Reference:

1. ENCODE at NHGRI
2. Press release from NHGRI: New Findings Challenge Established Views on Human Genome ENCODE Research Consortium Uncovers Surprises Related to Organization and Function of Human Genetic Blueprint 13 June 2007
3. Image courtesy: Pillars of Creation: Eagle nebula M16 by NASA Accessed 6-Aug-2007
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Gal1 gene silencing in Hodgkins's lymphoma

Margaret Shipp and team at Dana-Farber Cancer Institute (DFCI) have published novel target for Hodgkins' lymphoma (HL) and possibly other malignant tumor gene targeted therapy. In a study published in PNAS, Shipp et al. showed overexpression of specific protein Gal1 or Galectin 1 in pathognomic Reed-Sternberg cells of HL, at least 30 times higher than in normal cells. Galectin 1 showed to induce apoptosis and inhibit Th1 cells - a type of regulatory helper T-lymphocytes which recognize and kill foreign infectious agents and sometimes tumors. Relative increase in other helper T-cells, namely Th2 and Treg cells promote tumor survival and growth by inhibiting immune response to altered (tumor) cells by Th1 cells. Team documented reversal of Th1 activity and increase in Th1 cell count following RNAi mediated gene silencing of Gal1 gene. Earlier animal experiment by other team also showed possible role of Gal1 silencing in treatment of malignant melanoma. DFCI team is now developing targeted therapy to to counteract Gal1 induced tumor survival and hope to get fast-track experimental approval for clinical trials like other immunomodulatory agents..

Original article:
Margaret A. Shipp, et al. | The AP1-dependent secretion of galectin-1 by Reed–Sternberg cells fosters immune privilege in classical Hodgkin lymphoma | Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0706017104 (ahead of print publication) 1-Aug-07

Reference:
1. Press release from DFCI (30 july 2007)

Reprogramming the Cell | Part 4: iPS - Introducing next generations

Stem cells as a potential therapy for Diabetes, Parkinson's disease, Alzheimer's and many more - Ever since birth of the Dolly (1996) ¹, this line has became a hope for millions of victims of these high-burden diseases. However, stem cell research is often surrounded in ethical controversy which hindered its applied research to target potentially curable diseases. With the recent advancement in stem cell science and reprogramming the adult cell, scientists are now more optimistic than ever before to translate stem cell research on the clinical desk. Here is the remaining story of stop press article, posted on June 8.

Mature somatic unipotent adult cell can be reprogrammed to pluripotent form, known as induced Pluripotent Cell (iPS). Currently used methods, somatic cell nucleus transfer (SCNT, i.e.: Dolly) and cell fusion to cultivate embryonic stem cells (ESC) and cloning purpose are surrounded by ethical issues. Stem cells (i.e. blood stem cell) currently produced using SCNT (for autologus cell transplants and other therapies) lack long-term self-renewal capacity. Also, use of ESC poses increase chances of immune rejection compare to using patient's own adult cells. Overriding these hurdles, scientists are now exploring possible indirect ways to reprogram the adult cell back to embryonic state by manipulating signal pathways and genes related to cell differentiation using epigenetic reprogramming. Experimental animal studies in mouse revealed several critical genes related to pluripotency power of ESC, namely OCT3/4 (Pou5f1), SOX2, c-Myc and KLF4. ² These four transcription factors are involved in maintaining pluripotency (self-renewal) and upon subsequent cell differentiation of (fertilized) ESC, these factors are found to be silent by mechanisms yet to be identified.

The first generation of iPS was produced from mouse fibroblasts in 2006 by Takahashi and Yamanaka ³ using retroviral mediated transduction of these four factors and then selecting resulting cells expressing specific marker protein coded by Fbx15 ² thought to be marker of ESC pluripotency. This procedure did not require use of ESC at all for production of iPS cells (given name, Fbx15 iPS cells) which are similar to ESC in morphology, proliferation and teratoma (differentiation property) formation. However, subsequent gene expression and DNA methylation patterns of Fbx15 iPS cells were different from ESC and they fail to produce viable chimeras (offspring) indicating alternative pathways linked with cell differentiation or selecting incorrect marker of expression. i.e. Fbx15 in this case.

Within six months (in June 2007), same group published a breakthrough study along with two other independent research groups making successful reprogramming of mouse fibroblasts into iPS and even producing viable chimera. Maherali's team also documented reactivation of somatically silenced X chromosome in female iPS using same reprogramming method. ^4,5,6 This time, they used different selection marker, Nanog (from 'Tir Na Nog, the mythological Celtic land of the ever young) instead of Fbx15. Second generation of iPS is emerged as outcomes of all studies showed successful conversion of mouse fibroblast in iPS (now, named Nanog iPS cells) which are more similar to ESC in terms of gene expression, DNA methylation patterns and producing viable chimeras (and thereby contributing to subsequent germ-line production) indicating Nanog is a major determinant of cellular pluripotency. However, researchers also showed following hurdles before effective iPS can be substituted for ESC.

1. Nanog iPS cells have strong silencing of retrovirally expressed OCT3/4 (Pou5f1), SOX2, c-Myc and KLF4 c-Myc, indicating they are only required for the induction, but not the maintenance of pluripotency (pluripotency is then maintained by endogenous activated Oct3/4 and other genes in Nanog iPS cells). Yamanaka's team reported tumor development in approx. 20% of viable offspring which was attributable to reactivation of c-Myc through retrovirus activity. Since these factors are only required for induction of iPS synthesis, research is now focusing on using transient expression methods (i.e. using host-friendly Adeno-Associated Virus delivery of genes) rather retroviral induction of transcription factors.

2. Efficiency of Nanog cell induction is just 0.1% which points towards additional genes and epigenetic events required for successful formation of iPS.
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Although not precisely labeled as third generation of iPS, recently published paper documenting first ever successful parthenogenetic (meaning reproduction without sexual act or need of mating partner) activation of human oocytes and production of parthenogenetic human ESC (phESC) may be an another method to produce MHC matching cells for patient-specific cell-based therapy and minimizing immune rejection problem associated with ESC or SCNT. ⁷ Parthenogenetic stem cells are produced from unfertilized oocytes and contain genetic material 'exclusively' from the oocyte donor (the potential patient). Further research analysis is ongoing till this simple approach (in terms of procedure and equipment wise) may open new doors in treating degenerative diseases.

Related posts on Reprogramming the Cell
1. Part 1: Basics of Stem Cell
2. Part 2: Road to a unipotent adult cell (Programming Cell)
3. Part 3: Stop press

References:
1. Wilmut I, et. al. | Sheep cloned by nuclear transfer from a cultured cell line | Nature 1996;380:64-6 | PMID: 8598906
2. NCBI OMIM database: Oct3/4 (Pou5f1) | SOX2 | c-Myc | KLF4 | Fbx15 | Nanog
3. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126:663–676 | PMID: 17154061
4. Yamanaka S, et. al. | Generation of germline-competent induced pluripotent stem cells | Nature 2007;448:313-7 | PMID: 17554338
5. Wernig M, et. al. | In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state | Nature 2007;448:318-24 | PMID: 17554336
6. Maherali N, et. al. | Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution | Cell Stem Cell 2007;1:55–70
7. Revazova ES, et. al. | Patient-Specific Stem Cell Lines Derived from Human Parthenogenetic Blastocysts | Cloning Stem Cells. 2007 Jun 26; [Epub ahead of print] | PMID: 17594198 | Crossref.: Activated eggs offer route to stem cells- News@Nature
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Oncolytic Therapy in Cancer Management: An Emerging Approach

Oncolytic therapy using iatrogenic virus infection is a topic of extensive research for more than two decades and now series of clinical trials are being run in phase 1/2 stage which may bring hope to millions of cancer patients for better prognosis. Oncolytic virus is typically a host-friendly non-pathogenic virus which can preferentially infect cancer cells, leaving normal cells unharmed either because of its intrinsic nature or iatrogenic (designed in laboratory) manipulation.

Two most commonly researched viruses are Adenovirus and Herpes Simplex Virus (HSV) considering their latent to minimal clinical course, ability to self-replicate and stable genome structure (dsDNA). Similarly, virulent strains can be made non-pathogenic by creating their mutants in-vitro which are specifically toxic to abnormal tumor cells but not to normal cells. i.e. PV1(RIPO) mutant of Polio virus was made (Gromeier et al., 2000) to attack selective malignant glioma cells by replacing internal ribosomal entry site (IRES) of polio virus with non-pathogenic rhinovirus IRES.
Such tumor cell selectivity can be achieved by 1) altering virus surface proteins (transductional approach) using cDNA mediated gene expression/repression, modifying their protein structure and/or attaching surface proteins with fusion proteins (antibody to host-cell surface receptor) facilitating transport of virus in the cancer cell through cell surface. Another approach 2) is by non-transductional mechanisms which alter viral genome (transcriptional expression, deletion of genes) allowing virus to replicate within cell and thus, exacerbate cytotoxicity; introducing suicidal genes which facilitate adjuvant drug's actions (i.e. HSV thymidine kinase and ganciclovir) or suppressing angiogenesis genes to halt blood supply to tumor cells. Apart from altering viral genome, virus can also replicate to many folds leading to bursting of cancer cell and eventual death, known as viral cytotoxicity. i.e. Vesicular stomatitis virus (VSV) which is usually non-pathogenic can multiply to nearly 1000 folds in just 24 hr in cancer cells which lacks interferon-linked anti-virus defense machinery.

Oncolytic therapy holds one of promising innovative cancer therapy, especially taking consideration of recent advancement in molecular science and genetics. Here is list of selected upcoming agents of clinical interest. ¹

• ONYX-015 (CI-1042), an adenovirus modified selectively to replicate in and kill cells that harbor p53 mutations by deletion of E1B gene of virus which normally inactivates p53 protein. Such alternation leave normal cells unaffected while virus replicates and kills cancer cells having p53 mutation (which is the most common type of genetic abnormality in cancer, in almost 50% cases). Drug showed favorable results in head and neck cancers and went till phase 3 clinical trials in US and even commercial approval in China (Onyx-015/H101). However, further trials and production was suspended (Jan 2003) by Onyx pharma till further update. ^{2, 3}

• OncoVex GM-CSF product (phase 2 stage in melanoma) contain added gene that encode human GM-CSF which is a potent stimulator of immune system cells such as dendritic cells, proven to play role in cancer treatment. ⁴

• Cell Genesys's CG7870, Prostate-Specific Antigen (PSA) - targeted oncolytic Adenovirus therapy for prostate cancer, has been evaluated in Phase 1/2 clinical trials in combination with chemo-/radio therapy. ⁵ It uses PSA tumour-specific promoter sequence to promote expression of the adenoviral E1A gene, essential for replication.

• Respiratory Enteric Orphan virus (Reovirus), an inhabit of respiratory and intestinal systems is capable of replicating in tumour cells lacking the activity of PKR (anti-viral defense mechanism patent in normal cells) which is commonly seen in cancers having an activated Ras pathway. ⁶ Ras signal pathway is believed to play key role in more than two third of all human cancers. Oncolytic's Reolysin® (Reovirus oncolytic therapy) in combination with Docetaxel is just entered (23 July) in phase 2 clinical trial for advanced cases of bladder, prostate, lung and upper gastro-intestinal. ⁷
  

References:
1. Detailed information and reference links at Wikipedia article: Oncolytic Virus
2. Crompton AM, Kirn DH | From ONYX-015 to armed vaccinia viruses: the education and evolution of oncolytic virus development | Curr Cancer Drug Targets 2007;7:133-139
3. Onyx Pharma Press release: Onyx Increases Development Focus on Bay 43-9006 | Jan 27, 2003
4. Biovex's OncoVex: Product page
5. Cell Genesys's CG7870: Product page
6. Article from hospitalpharma.com
7. Oncolytics Pharma Press release: Oncolytics Biotech Inc. Starts Patient Enrolment in U.K. Combination REOLYSIN®/Docetaxel trial
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Cancer: Spark and Shoot by painting

Researchers at Seattle Children’s Hospital Research Institute and Fred Hutchinson Cancer Research Center have developed innovative method to demarcate tumor cells in "live" operating room from surrounding healthy cells by illuminating tumor cells with fluorescent material, Cy 5.5 (Cyanine) which can be detected by operating surgeon using infrared capable glass/surgical microscope. In an animal study, trial was done on mouse models having glioma . Upon injecting Chlorotoxin:Cy5.5 (where Chlorotoxin is a scorpion derived peptide, currently in clinical trials as a anti-cancer therapy which specifically binds to the surface of glioma cells and impairs their ability to invade), cancer cells as small as 1 millimeter in diameter took up this biconjugate agent and emitted light which was captured by infrared device. This will boost accuracy and safety of surgical approach in managing brain tumors and many others in near future.

This technology claimed very high sensitivity with comparison to currently used MRI of brain with contrast agent. According to team member, Olson JM, MD: This is particularly significant in the brain, where approximately 80% of malignant cancers recur at the edges of the surgical site. MRI can distinguish tumors from healthy tissue only if more than 1 million cancer cells are present. But Cy5.5 can identify tumors with as few as 2000 cancer cells, making it 500 times more sensitive than MRI.

Olson: Chlorotoxin:Cy5.5 could be used in operating rooms in as little as 18 months.

Team has also conducted trial in mouse models having prostate cancer and found favorable results. After injecting Chlorotoxin:Cy5.5, it begins to bind cancer cells in hours and signal lasts for 14 days compare to rapid elimination within minutes of currently used contrast agents in radiology. This can be a promising non-invasive screening tool for early detection of skin, cervical, esophageal, colon and lung cancers as well as identifying positive lymph nodes critical for breast, prostate and testicular cancers management and follow-up. Team is planning to deploy phase 1 clinical trial soon.

• Original article: Mandana Veiseh et al. | Tumor Paint: a chlorotoxin:Cy5.5 bioconjugate for intra-operataive visualization of cancer foci | Cancer Research 2007:67 (scheduled for release on July 15, 2007)

Reference:
1. EurelAlert! 15 July 2007 release
2. Deshane J, Garner CC, et al. | Chlorotoxin Inhibits Glioma Cell Invasion via Matrix Metalloproteinase-2 | J Biol Chem 2003;278:4135-4144