I had written this article two semesters back. Basically, it talk about this new technology called induced pluripotent (iPS) cells. These can be made from adult somatic cells. Basically, the iPS cells are almost exactly like the immensely controversial embryonic stem cells. This may herald the end of the stem cell controversy because iPS cells are made by expressing 4 genes within the cells. This means no embryos are harmed in their construction. Hopefully, this technology will help US scientists come up to speed with the rest of the world in terms of stem cell work. It may also be another step closer to the cure for neurodegenerative diseases such as Parkinson's and ALzheimer's.
The horse changed its mind about jumping over the fence and the burly rider was thrown violently to the ground. The helmet saved his brains, but the impact shattered the first and the second vertebrae; His spinal cord was disconnected from his brain. Unlike skin cells that are replaced everyday, cells of the nervous system don’t mend themselves once damaged. Superman doesn’t fly off-screen, after all.
Christopher Reeves went on to campaign for Human Embryonic Stem Cell research. His death in 2004 didn’t decelerate the race to develop stem cell technology to a point where a miracle cure for people like him would exist. In addition, stem cell mediated regenerative therapies have frequently been considered the way to cure degenerative diseases such as Alzheimer’s disease, Parkinson’s disease, etc.
Recently, some exciting events have taken place in the world of stem cells. This review paper recounts some of the recent advances in this marvellously promising, but frightfully controversial, field of science. Indeed, The Economist has dubbed this field “the most operatic in science.” However, it is imperative to establish some basic facts regarding stem cells first.
Stem cells are pluripotent cells; undifferentiated cells that have the potential to become any cell type. They can be classified into two types: adult stem cells and embryonic stem cells. Adult stem cells are used to repair and replace cells that are lost during wear and tear. These can be found in minute quantities within the bone marrow and the umbilical cord. Embryonic stem (ES) cells have the potential to mature into any cell type. They are collected from the inner cell mass of embryos in the blastocyst or morula stage of development. ES cells have significant potential in regenerative therapies.
However, it is distinctly possible for the body of a stem cell recipient to reject it due to histo-compatibility issues. Therefore, it was proposed to prepare embryonic stem cells using the
Unfortunately, stem cells can’t be extracted from embryos without destroying the latter. This aspect of stem cell research offends the moral sensibilities of those who oppose it. Political ideologues have deftly entangled the stem cell extraction process with the abortion controversy. Consequently, the moral outrage of a minority of tax-payers has brought stem cell research in the
The success of SCNT and the subsequent cloning of Dolly4 made the scientific community aware of the existence of cytoplasmic trans-acting factors that were capable of reprogramming somatic cells into reverting to an embryonic stem cell like state. This realisation prompted a hunt for these transcription factors.
In November 2007 --10 years after Wilmut’s critical breakthrough-- Takahashi et al. reported their success in synthesising pluripotent cells without using the controversial SCNT protocol. They reported that induced pluripotent cells (iPS) could be produced from human dermal fibroblasts (HDF) by transducing four transcription factors- Oct3/4, Sox2, Klf4, and c-Myc.1
It seems almost amazing that the transduction of four transcription factors can lead to such an amazing transition from an HDF to a pluripotent cell. Prior to Takahashi et al., Wernig et al. had shown that Oct3/4, Sox2, Klf4, and c-Myc can epigenetically reprogramme a mouse somatic cell into an embryonic stem cell like state2. These seemed to be some of the aforementioned trans-acting factors involved in converting somatic cells into embryonic stem cells. Oct3/4 and Sox2 have been shown by previous studies to act as the central pluripotency generating transcription factors. Takahashi et al. speculate that c-Myc and Klf4 make the chromatin structure more conducive to the binding of Oct3/4 and Sox2. This speculation is strengthened by the fact that Klf4 is known to regulate the acetylation of histones1.
Wernig et al. transduced the four transcription factors into mouse fibroblast cells. iPS cells were selected for by looking for the activation of a gene downstream of Oct4- Fbx15. However, the cells, though pluripotent, were dissimilar to ES cells in some regards. For instance, some differences exist in the methylation patterns and gene expressions of ES cells and iPS cells selected using Fbx15. Furthermore, these iPS cells do not contribute to viable chimaeras2.
Okita et al. showed that selecting for Nanog instead of Fbx 15 produces cells that resemble the ES cells even more with regard to epigenetics and gene expression3. Nanog is more closely associated with pluripotency than Fbx 15 since it is known that disrupting Nanog in mice results in a loss of epiblast pluripotency. Conversely, disrupting Fbx15 doesn’t produce a readily observable effect. The researchers, isolated a bacterial artificial chromosome (
The ES cells that stably incorporated the GFP were, then, introduced into mouse blastocysts to obtain chimaeric mice that were used to produce transgenic mice containing the aforementioned reporter construct (Nanog-GFP-IRES-Puror). The blastocysts from the transgenic mice contained the reporter in the inner cell mass. The primordial germ cells were found to be GFP-positive 9.5 days post coitum (d.pc.) and by the 13.5 d.p.c, the genital ridges were also GFP positive.
Okita et al. extracted MEF (mouse embryonic fibroblasts) cells (which were GFP negative) and introduced the aforementioned transcription factors into the mouse cells using retroviral vectors. The researchers used a mutant of c-Myc (T58A) instead of the wt gene. The MEF cells were cultured on feeder cells. After giving the cells time to heal from the retroviral infection, puromycin selection was commenced. Twelve days post retroviral infection, hundreds of GFP positive colonies were observed. Some GFP-negative colonies were also present. The researchers are unsure about the reason for their occurrence. By contrast, no GFP positive colonies were observed when mock
Bisulfite sequencing was used to show a congruence between Nanog selected iPS cells and ES cells in their methylation patterns. Methylation patterns reveal the pattern of gene silencing in a genome.
Furthermore, these iPS cells resembled ES cells in their morphology (ES cells have flat colonies), teratoma formation, and proliferation pattern. It was shown that Nanog gets downregulated when a cell begins to differentiate, thus further strengthening the case for using Nanog as a selection criterion for pluripotent cells. However, the induction efficiency was very low. Now that the induction of pluripotency using these four transcription factors has been established, it would be profitable to scrutinize the work of Takahashi et al. in greater detail.
The first step taken was to increase the transduction efficiency of the HDFs they were working with. This was achieved by the introduction of a mouse retroviral receptor (Slc7a1) using a lentivirus as a vector. These HDF-Slc7a1 cells had increased transduction efficiency from 20% to 60%. This was assayed by using a gene construct with GFP.
Subsequently, retroviruses containing Oct3/4, Sox2, Klf4, and c-Myc were introduced into the HDF-Slc7a1 cells. Six days after transduction, the cells were harvested and plated onto feeder cells. The next day the medium was replaced with a medium for primate embryonic stem (ES) cell culture (supplemented with basic fibroblast growth factor). At first, granulated colonies that did not resemble ES cell colonies were seen. However, by the 25th day after transduction, some flat ES cell-like colonies were seen. They were called human induced Pluripotent (iPS) Cells.1
It was found that the human iPS cells resemble human ES cells in numerous ways. Takashi et al. carried out reverse transcriptase polymerase chain reaction analysis (RT-
Moreover, western blot analysis reported that the iPS cells resembled ES cells in their OCT3/4, SOX2, NANOG, SALL4, E-CAD-HERIN, and hTERT protein levels. Western blot is a technique used to assay the expression of a specific protein. Essentially, the protein is extracted and electrophorosed. After the electrophoresis, the proteins are transferred onto a nitrocellulose (or PVDF) membrane where they are probed with antibodies. Subsequently, a secondary antibody is added. This antibody attaches to the primary antibody. Usually, the secondary antibody is fluorescent or chemo-luminescent. By monitoring the abundance of the secondary antibody, it is possible to estimate the level of protein expression. By comparing the displacement of the band to a molecular marker, it becomes possible to identify the protein on the basis of its mass.
Proteinaceous trans-acting factors bind to specific DNA domains. However, they don’t bind to all possible domains at the same time. It is possible to infer DNA methylation and gene silencing patters based on information regarding the DNA sequences that a particular regulatory is binding to in a cell. This can be done using ChIP analysis. Basically, the DNA is extracted and broken down mechanically. Subsequently, the broken up DNA is probed with the antibody of the regulatory protein. This yields a collection of DNA sites that the regulatory protein was bound to. This methodology can be, and was, employed to detect histones modified by methylation. The ChIP analysis showed that the histones in the promoter sites of Oct 3/4, Sox2, and Nanog were demethylated (unlike the HDF cells that were highly methylated at these loci).
In addition, iPS resembles ES cells in their high telomerase activity, exponential proliferation, and teratoma formation. Furthermore, Takahashi et al. were also able to demonstrate the ability of the iPS cells to differentiate into dopaminergic neurons by co-culturing them with PA6 cells. They demonstrated using
This characteristic of the iPS cells creates hope for people suffering from degenerative diseases such as Parkinson’s and Alzheimer’s. Even more phenomenally, the iPS cells were induced, using Activin A and bone morphogenic protein (BMP), to turn (in-vitro) into cardiomyocytes. Within 12 days of induction, the cells began beating. RT-
In addition to the HDF cells (taken from a 36 year old Caucasian woman) that were converted into iPS, human-fibroblast-like-synovocytes (HFLS) from the synovial joint of a 69 ear old man were also successfully turned into iPS cells. For an encore, Takahashi et al. also turned BJ cells from neonate fibroblasts into iPS cells. All the iPS cells resembled the HDF iPS cells in the aforementioned ways.
There is, however, a small drawback to this procedure. It was found that each iPS clone receives three to six retroviral integrations per transcription factor. This increases the probability of tumour formation. Indeed, 20% of mice derived from iPS cells possessed tumours. This has been blamed on the reactivation of the c-Myc retrovirus1. c-Myc is a known oncogene. Indubitably, the utilization of c-Myc is a risky proposition. The usage of retroviruses is controversial as well and the authors propose to either begin using adenoviruses instead, or to find molecules small enough to induce gene transfer sans gene transfer.
However, it seems the, somewhat hazardous, transduction of –Myc is not imperative for the creation of iPS cells. Yu et al. made a list of genes that were enriched in ES cells relative to myeloid precursors. OCT 4 is known to be expressed greatly in pluripotent cells and is also known to confer geneticin resistance to cells. Hence, geneticin selection was used to isolate pluripotent cells. Initially, a combination of 14 genes was used to reprogramme CD45+ haematopoetic cells into OCT4 positive, geneticin resistant cells that resembled ES cells in the morphology and cells surface markers. Re-testing was carried out. Finally, using this approach, Yu et al. showed that OCT4, SOX2, NANOG, and
In other news, Byrne et al. report the production of primate ES-cells using a modified SCNT protocol. Byrne had previously reported that the removal of lamin A/C was essential for the remodeling of the oocyte into an ES cell. This required the functioning of a maturation-promoting factor (MPF) which might have been degraded in the previous protocol (due to the usage of Hoechst stains and UV light) resulting in non-removal of lamin A/C. The old protocol also tended to damage mitochondrial
Why should we care about all this? Stem cells are a fascinating resource. They can be used extensively for research purposes. For instance, if it is difficult to obtain diseased tissues to study, patient-specific cells can be concocted to study the disease (using ES cells) since these cells are identical to the diseased cells. Drug trials could be expedited with the use of these iPS cells as well. Obviously, regenerative medicine is one of the most touted, as well as the most famous, applications of stem cells.
Some concerns do exist about the long time delays and the difficulties in producing the differentiated treatments required for regenerative medicine. However, one thing that has been established by these recent developments in the stem cell world is that technology and science are sprinting rapidly. It is very likely that in a very short amount of time, regenerative medicine will go from just being a distinct theoretical possibility to a routine medical procedure. As this paper was being finished, the media announced that a putative stem cell treatment for Duchenne’s muscular dystrophy has been developed. Excitingly, the DMD treatment technique can find applications in other genetic diseases as well. Things like neuronal regeneration are right around the corner. Superman flew away too soon.
1. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. (2007). Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. Cell 131, 1-12.
2. Wernig, M., Meissner, A., Foreman, R., Brambnik, T., Ku, M., Hochedlinger, K., Bernstein, B.E., and Jaenisch, R. (2007). In Vitro Reprogramming of fibroblasts into a pluripotent ES-cell like state. Nature 448, 318-329.
3. Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Generation of germline-competent induced pluripotent stem cells. Nature 448, 313-317.
4. Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KHS. (1997). Viable offspring derived from fetal and adult mammalian cells. Nature 385, 810-813.
5. Yu, J., Vodyanik, M.A., Smuga-Otto, K., Antosieqicz-Bourget, J., Frane, J.L., Tian, S., Nie, J., Jonsdottir, G.A., Ruotti, V., Stewart, R., Slukvin, I.I., and Thomson, J.A. (