IPS cells certainly solve the ethical dillema around use of ES cells, the next step will be to find ways to get high efficiency transfection with non viral vectors and figuring out ways to stop tumorigenesis.

http://www.nature.com/nrc/journal/v11/n4/pdf/nrc3034.pdf

Best

-Sardar Uddin

Clinical application, including safety administration

The translation of the iPSC technology to the bench side of patients should be focus once the technology is optimised.

I think the next step will be making this procedure safer in order to make iPS cells more identical to stem cells originated from blastocyst and after reaching to this step the next step will be its contribution toward curing diseases which are hard to cure.

Teaching the cells how to behave without reprogramming.

1. Drugs developed by using patient derived iPS cells for drug-screening will show up sooner or later.

2. Tissue or organ regeneration is promising but still a long way to go even with non-viral iPS generation technology, main difficulties: low efficiency of differentiation with contamination of undifferentiated cells, immune rejection (even using patient own cells), Survival of cell and connection of injected cell with host tissue in vivo.

Further cell tracking and monitoring micro-enviroment.

Before iPS can be applied in clinic, there remain many issues to be addressed, how to promote cell survival and directional differentiation under in vivo adverse local environment? how to direct functional reconstruction? and so on.

I believe that IPSC has opened up a new gateway with a sizable opportunities to be explored.

1) In vitro model for various diseases as in case of neurodevelopmental disorders.

2) Further understanding of epigenetic regulation of gene expression in stem cells.

3) In vitro model for cancer cells.

Possibilities are immense yet to be explored plus the ethical concern over ESC would no longer be barrier for stem cell research as to the use of IPS

The importance is the clinical application and iPS are the answers, but immunogenicity need to be addressed.

As many says before. It will be very important to generate iPS virus free, then immunogenicity is important and what also is important to get differentiated cells from these iPS cells which are the "same" as the naturally ones.

The fact that stem cells have become the focus of wordwide attention through the attribution of the Nobel Prize is highly laudable and will certainly help to channel additional resources towards this extremely important area of science and medicine. Yet, I reserve the privilege of dissent: Up to now, iPS are more the subject of fashion in many scientific circles, and have yet to prove their promises. I simply believe that we do not know enough (and never will) about the process of differentiation to be able to perform low-risk cell reprogramming at a given time in the future. In my opinion, other methods resp. cell sources (cord blood, autologous bone marrow) resp. Cell types (HSC, MSC, VSEL) will open up future paths, mainly in regenerative medicine.

Hello Thomas,

you are right. Here we discuss some month ago also the future of iPS using in medicine. And the conclusion was what you posted. We never will get cells for therapeutic use from iPS. This was also the reason why I write "same" - I think they will never be the same.

What is very nice from iPS cells and there differentated cells like nerve cells, cardiomyocytes etc. is that you have patient specific epigentic backround. This is very important to study diseases in many cases and can't do with other cell systems like HEK293 where you overexpress one protein and don't have the whole epigenetic backround of the patient.

Hi Sven,

thanks for your info. I'll look up the named discussion on the future of iPS.

Hi Thomas,

you find it here:

https://www.researchgate.net/post/Studying_disease_neural_tissue_in_vitro_using_iPSCs

Best,

Sven

What I realize after these postings is that the discussion mainly regards iPS, whereas I was thinking that it will be more useful to get to learn more from the adult stem cells from each tissue and what controls their differentiation. Would not this make more sense for any possible therapeutical use?

I think it is important to know every thing about adult stem cell. The whole biology of these cells I think is not yet completed. If we know the whole detail ,this may help in exploring iPSC. Going from lab experimentation to human therapy is something we need ,but we have to be real cautious in implementing it. Rush to human therapy may be dangers .We have a lot of auquard situations before in applying other advanced tech

Jose, whenever you hear "stem cells", you must always ask "WHICH stem cells?" Induced pluripotent cells are not all of "stem cell biology". In fact, they are only a very small part of it. As several people have pointed out, iPS cells have some major problems, with the top 2 being 1) one of the genes that has to be introduced is an oncogene and 2) they form teratomas when implanted in a mass. So what people are trying to do is manipulating iPS cells to differentiate them before they are put in the body. That adds cost and more safety concerns because they use viral vectors to transduce for the differentiating genes.

Meanwhile, many researchers are working on various adult stem cells. Some have the same differentiation potential as the iPS cells without the safety concerns. One adult stem cell -- the mesenchymal stem cell from Osiris -- has been approved in 3 countries for treatment of Crohn's disease in children. Other adult stem cells are in phase I and II clinical trials. This puts them far ahead of the iPS cells in terms of clinical use.

So, what is next in stem cell biology may very well be adult stem cells instead of iPS cells.

Dear Paul,

thanks for your comments, I fully agree with you, and when I asked the question, I was referring to Stem Cells, rather than to iPS, although most of the comments have been about the latter.

With regard to MSC the main problem I have is that although they are used in clinical trials as you point out, unfortunately these are one the less well defined adult stem cell population (although I also do some work with them). I believe that the cells from the intestine, mammary gland, brain are better defined than MSC....

Jose:

It depends on what you mean by "well defined". Different stem cells have different differentiation potential. The stem cells from the intestine, for instance, can only differentiate to the epithelial cells lining the small intestine (unipotent). They do have a definitive marker, but they are not going to be very interesting clinically. One reason is that you have to rob Peter to pay Paul. That is, you need to harvest the intestinal stem cell from one part of the intestine (destroying that part of the intestine) to get the stem cells to repair another section of the intestine. Neural stem cells from brain also pose a problem clinically: how do you get a biopsy of brain to isolate the cells? Who is going to let you go in and cut out a chunk of their brain?

MSCs are "well-defined" in that they can be isolated by being positive for 3 CD markers: CD 105, CD117, and CD166. If a cell is positive for all 3 of these, then it is an MSC. They are much more interesting clinically because they have such a varied differentiation potential (multipotent) and they are rather easily isolated. They can be isolated from bone marrow aspirates but used to potentiatly regulate the immune response (Crohn's disease), regenerate bone, regenerate cartilage, provide scaffolding for new marrow after radiation therapy (in leukemia), etc. What is more, they lack MHC-I, which means allogenic cells can be used.

The downside to MSCs are 1) their site of harvest and 2) their limited proliferation potential. There is significant morbidity associated with doing a marrow aspirate. The MSCs obey Hayflick's limit, which means that they have a limited number of cell divisions before they senesce and die. This is why Lonza can sell them for research purposes. Osiris says they can treat 10,000 patients from one marrow donor, but this is done with a very limited number of MSCs. Some applications, such as articular cartilage repair, are going to require hundreds of millions of MSCs, and there simply are not enough. If the small number implanted expand to this number, then they will reach replicative sensescence and die, thus destroying the implant. Replicative senescence is what happened to Carticell and why it is limited to treating people under 30: the cultured chondrocytes of older patients died in vivo after a few years.

Most people who work with MSCs do not do the cell sorting by FACS for the 3 CD markers. Instead, they take the adherent cell population from marrow. This is indeed undefined. There are osteoprogenitor cells, adipocytes, fibroblasts, smooth muscle cells, MSCs, and at least 2 other types of multipotent adult stem cells: Verfaillie's MAPCs and Lucas' MASCs.

Jose: "I was thinking that it will be more useful to get to learn more from the adult stem cells from each tissue and what controls their differentiation. Would not this make more sense for any possible therapeutical use? "

The adult stem cells you refer to are "progenitor stem cells" or "tissue specific stem cells". Yes, they have the ability to differentiate to the cells of that tissue, but ONLY that tissue. They are "unipotent". They also have a limited proliferation potential, which means the number of cells you can generate is limited. A classic example of this is the muscle satellite cell and Duchenne's muscular dystrophy. The satellite cell is a mononuclear cell that resides between the cell membrane and the basal lamina of skeletal muscle. Skeletal muscle cells themselves are multinucleated with hundreds/thousands of nuclei and are very long. They run the entire length of the muscle. So the skeletal muscle cells in your biceps runs from near the origin in your shoulder to the insertion in your forearm.

Anyway, under normal circumstances the satellite cells divide and the daughter cell becomes incorporated into the myotube (multinucleated skeletal muscle cell). This maintains the skeletal muscle. In muscular dystrophe (MD), the myotubes do not produce a functional dystrophin protein and this leads to the disintegration of the myotube. The satellite cells then multiply and regenerate the myotube. But the new one also lacks dystrophin (since it is an inherited genetic defect) so the new myotube also disintegrates. And again the satellite cells divide and regenerate the myotube. The cycle keeps going until the satellite cells reach the end of their proliferation potential and cannot divide anymore. At this point the satellite cells senesce and die and the muscle is replaced by fibrous tissue. In Duchenne's MD patients, this happens first usually in the diaphragm somewhere in the early 20s, and without the diaphragm, the patient cannot breathes and expires.

So, the progenitor stem cells have 3 things that limit their clinical use: limited proliferation potential, limited utility, and limited numbers. However, the body contains more primitive stem cells that have wider differentiation potential and extended proliferation. The hematopoietic stem cell (which can form any of the blood cells) is one of these. Their differentiation is multipotent but limited to blood cells. Even so, they have great therapeutic use in treating leukemias: kill off all the patient's hematopoietic cells (including the leukemic cells) and then repopulate with hematopoietic stem cells to make new blood.

Neural stem cells are multipotent in making all the cell types in the brain: neurons, astrocytes, and oligodendrocytes. But taking brain tissue to isolate the neural stem cells is not a real clinical option.

Research tends to focus on multipotent adult stem cells with extended proliferation potential that can come from sites where it is easy and (relatively safe) to obtain tissue: MSCs from bone marrow, Adipocyte Derived Stem Cells from fat, Endometrial Regenerative Cells from discarded endometrium of the uterus, Multipotent Adult Progenitor Cells from bone marrow, etc.

Thanks Paul.

yes I meant adult stem cells. Another example of multipotent are the mammary stem cells. So far in vitro have strict asymmetric divisions, but in vivo also expand (20-fold during puberty), and can reconstitute a functional mammary gland on serial transplants (6, 7,...)

Dear Jose,

I would agree with Paul Lucas. But I would also look at the adult-derived pluripotent stem cells and the adult-derived totipotent stem cells, both of which are found in any organ with a connective tissue component. And both express the telomerase enzyme, which gives them extended capabilities for self renewal well past Hayflick's limit for dfferentiated cells or progenitor cells.

Scale-up is also a major concern moving from the routine laboratory animals (mice, rats, and rabbits) to human clinical trials. We found that it is far better to examine a similar problem in porcine, bovine, and/or equine animal models in preclinical trials to determine all parameters possible, than to jump ahead to clinical trials in humans. And given the size of these larger animal species, we pretty much don't need to worry about scale-up once we have defined all the parameters.

Jose, if by "mammary stem cell" you mean the cells discussed here -- http://jcs.biologists.org/content/118/16/3585.full -- then yes, they are "multipotent" in that they can form at least 3 phenotypes. This is where the nomenclature of stem cells leaves something to be desired. In this case, all the phenotypes are within a single organ: the mammary gland. In this regard they are similar to the multipotentcy of hematopoietic stem cells: all the phenotypes of blood.

However, "multipotent" is also used to refer to stem cells that have differentiation capability outside a particular organ. Back to our old chestnut the mesenchymal stem cell: it can differentiate to cells of bone, cartilage, muscle, or fat. All those are different organs. Bjornsen et al. have a paper describing neural stem cells that differentiate into blood cells: http://www.ncbi.nlm.nih.gov/pubmed/9915700 Catharine Verfaillie describes cells from bone marrow that can not only differentiate into the same phenotypes as mesenchymal stem cells, but also hepatocytes: Journal of Clinical Investigation, 109: 1291, 2002. She calls these cells Multipotent Adult Progenitor Cells. My group has a paper -- http://www.ncbi.nlm.nih.gov/pubmed/16630113 -- describing adult stem cells that can differentiate into phenotypes from many different organs. We call them "Multipotent Adult Stem Cells". So here "multipotent" means something very different than it means for the mammary stem cells.

What needs to be done is look past the name to what the cells are actually reported to be able to do -- which stem cells. :)

Dear Jose, Paul, and all

I have read some few literatures on the trans-differentiation potential of the haematopoietic stem cells in regenerating tissues like the liver after injury. Are these submissions robust? Or have you had any such experience/findings with the haematopoietic stem cells?

The situation is complicated -- what a surprise. First, there seems to be a problem identifying "the" hematopoietic stem cells. Some groups claim it is CD34+, others that it is CD34-. Others, such as Orlic, claim a different population with different markers.

Second, there are some recent papers out calling into question the differentiation of hematopoietic stem cells into hepatocytes: http://informahealthcare.com/doi/abs/10.3109/08941939.2011.627088

J Invest Surg. 2012 Aug;25(4):220-6. Epub 2012 May 9. Influence of hematopoietic stem cell-derived hepatocytes on liver regeneration after sex-mismatched liver transplantation in humans. Schwerfeld-Bohr J, Chi H, Worm K, Dahmen U.

http://www.ncbi.nlm.nih.gov/pubmed/23112081

http://www.ncbi.nlm.nih.gov/pubmed/23034562

So it looks like the answer to your question is that this is an area of controversy. I think a statement from the last reference sums it up: "Bone marrow-derived hematopoietic stem cells (BM-HSCs) have been shown to act as source for hepatic regeneration in rodent models; however, their ability to participate in human liver regeneration remains controversial."

I personally do not work with hematopoietic stem cells, but I have serious doubts that the hematopoietic stem cell has any differentiation potential beyond hematopoietic cells. I also have to say that I think the term "transdifferentiation" is inaccurate. I don't think cells transdifferentiate to other lineages. Instead, I think the data shows that there are primitive stem cells capable of differentiating into a number of phenotypic lineages. However, once a cell is committed to a lineage -- say an osteoblastic lineage -- then it cannot "transdifferentiate" to a different lineage, say an adipocyte lineage.

Generation of artificial tissues and organs from stem cell differentiation will be one of the exciting fields in stem cell research in years to come.

As hype will go down about the use of stem cells in tissue regeneration and reparation the next thing will be heavy hangover followed by the next hype about something followed by next Nobel Price. The next thing, in my opinion, will be reprogramming organ/tissue microenvironment toward healthy signaling pattern as opposite to diseased organ/tissue. Induced pluripotent stem cells are unlikely to be a part of it. Adult stem cells a likely candidates. However, realization of this will require drastic modification a pattern of thoughts, especially by industry.

:)

I completely agree with you, Sergey.

Field of Mesenchymal stem cells is also gaining therapeutic advantages over various clinical indications. And MSCs, in the clinical arena to treat different diseases is advancing rapidly, as reflected by the increasing number of publications and clinical trials.

Dear Patrick,

I fully agree with Paul Lucas' reply. On the other side, if you are able to generate an ES-like cell (iPS) from a fibroblast, you may think that transdifferentiation is possible. Similar findings have been reported from neurones able to generate neural precursors, or more recently Robert Weingerg'lab has shown that expression of SOX9 and slug on differentiated epithelial cells allow the generation of Mammary Stem Cells (if you over express only SOX9 you get luminal progenitors, while the cells expressing only SLUG are Myoepitelial cells and Myoepithelial progenitors).

From there to trans-differentiation there is a simple step. The only problem is to show that it is bona-fide transdifferentiation, which is not easy (you should discard cell fusions, etc)

Mesenchymal Stem Cells with immunosuppressive, immunomodulatory and proactive paracrine properties makes them remarkable candidates for specific targets in a disease.The property of these cells to adapt to the damage niche and act accordingly and have no allo-reaction have made them a safe and efficacius candidate in cell therapy.

I think the in stem cell biology the adult stem cells are easily obtainable cells and have lots of advantage over ESC and iPSCs regardign ethics, biosafety and teratoma formations. Among adult stem cells, the most easy source is adipose tissue. So the adipose derived mesenchymal stem cells have huge potential in clinical applications. I think it will boom in near future especially in regenerative medicine. The easy availability of soure, ease of isolation in clinically abundant amount with high viability, immune modulation, plasticity and ease of maintenance in the culture for longer period and all makes it very favourite candidate in regenrative medicine. There are few clinical trials are going on and lots of clinicl trials is going to come in future on this stem cells. It might hijack lion part of regenerative therapy in future!!!!

Dear Jose,

I think the challenges of iPS in the next 10 years would be dealing with the epigenetic memory of the reprogrammed somatic cells that might limit their potential for therapeutic use, or briefly to optimize the reprogramming process.

Nuclear transfer?

Cell, 15 May 2013

Copyright © 2013 Elsevier Inc. All rights reserved.

10.1016/j.cell.2013.05.006

Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer

Summary

Reprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state.

Dear,

Emerging evidence is shedding light on a large and complex network of epigenetic modifications at play in human stem cells. This “epigenetic landscape” governs the fine-tuning and precision of gene expression programs that define the molecular basis of stem cell pluripotency, differentiation and reprogramming. This review will focus on recent progress in our understanding of the processes that govern this landscape in stem cells, such as histone modification, DNA methylation, alterations of chromatin structure due to chromatin remodeling and non-coding RNA activity. Further investigation into stem cell epigenetics promises to provide novel advances in the diagnosis and treatment of a wide array of human diseases.

Actually those iPS induced tissues also have been rejected by host body in some case, that is a really big issue for its development.

Is there a certain mechanism or method to downregulate the expression of HLA-DR and co-stimulatory molecules on iPS?Small molecules maybe?If so there can be a possibility to evade the host immune system.

Hi charan if you see the recent paper from Prof. Jaenisch group they have tried to eliminate the class I HLA molecules on the ES cells using ZFNs. I think similar approach would be possible to eliminate HLA -DR also.

Towards eliminating HLA class I expression to generate universal cells from allogeneic donors.

Hiroki Torikai, Andreas Reik, Frank Soldner, Edus H Warren, Carrie Yuen, Yuanyue Zhou, Denise L Crossland, Helen Huls, Nicholas Littman, Ziying Zhang, Scott S Tykodi, Partow Kebriaei, Dean A Lee, Jeffrey C Miller, Edward J Rebar, Michael C Holmes, Rudolf Jaenisch, Richard E Champlin, Philip D Gregory, Laurence J N Cooper

Division of Pediatrics, The University of Texas MD Anderson Cancer Center, Houston, TX, United States;

Blood (impact factor: 9.9). 06/2013; DOI:10.1182/blood-2013-03-478255

Source: PubMed

ABSTRACT Long-term engraftment of allogeneic cells necessitates eluding immune-mediated rejection which is currently achieved by matching for human leukocyte antigen (HLA) expression, immunosuppression, and/or delivery of donor-derived cells to sanctuary sites. Genetic engineering provides an alternative approach to avoid clearance of cells that are recognized as "non-self" by the recipient. To this end, we developed designer zinc finger nucleases (ZFNs) and employed a "hit-and-run" approach to genetic editing for selective elimination of HLA expression. Electro-transfer of mRNA species coding for these engineered nucleases completely disrupted expression of HLA-A on human T-cells, including CD19-specific T-cells. The HLA-A(neg) T-cell pools can be enriched and evade lysis by HLA-restricted cytotoxic T-cell clones. Recognition by NK-cells of cells that had lost HLA expression was circumvented by enforced expression of non-classical HLA molecules. Furthermore, we demonstrate that ZFNs can eliminate HLA-A expression from embryonic stem cells which broadens the applicability of this strategy beyond infusing HLA-disparate immune cells. These findings establish that clinically-appealing cell types derived from donors with disparate HLA expression can be genetically edited to evade an immune response and provide a foundation whereby cells from one donor can be administered to multiple recipients.

The field of adult stem cells especially the mesenchymal stem cell; is gaining a lot of momentum in terms of the easily and readily availbility of the source, their profilerative and differentiation capabilities etc. I have been working with the adutl stem cells both Haematopoeitic and mesenchymal stem cells for almost a decade now and in terms of its clinical applications, both the cells hold a lot of potential. Actually both these cell types compliment each other therefore in certain clinical condition both these cell types in combination will help improving the clinical conditions. So the future is of adult stem cells!!! : both Mesenchymal stem cells and Haematopoeitic stem cells. Since these cell types can also be used in an allogeneic setting and hence the scope of these cells for patients in whom autologous cells is an issue.

So many unprecedented achievements have been made. Reprogramming technology certainly give us a promising further for regenerative medicine. However many obstacles still exist. For instance, most of reprogramming methods involved genetic manipulation whose safty are still concerned. Obviously, reprogramming by virtue of non-biological approaches should be an attractive direction. Encouragingly, Dr. Hongkui Deng in Peking University reported they latest discoveries that reprogramming mouse somatic cells into iPSCs by pure chemical compounds (published in Science DOI: 10.1126/science.1239278) which will open a much simpler and safer way for reprogramming. Furthermore, direct convertion of somatic cell into desired cells may be another hot trend. Additionally, reconstruction of tissues of organs with integrating stem cells and artificial scaffolds in 3D mode also hold great potentials for further regenerative medicine.

Thank yo Jose for posting this very intriguing question. I work on adult mammary stem cells under the premise that understanding the mechanisms that regulate their normal proliferation, differentiation, and self-renewal will help us understand how alterations to such fundamental mechanisms may lead to disease. I believe through this understanding, more specific therapies against cancer can be devised. More immediately however, I believe that understanding the mechanisms behind the normal functioning of the adult stem cells will yield better diagnostic biomarkers.

The discussions have been really thought provoking. Though, I do beleive iPS are at present most useful, it is easier and foolproof method to use adult stem cells if cultured without changes in their characteristics.

Dr.H. N. Madhavan.

I would think the further discoveries of various growth factors, differentiating factors, regulatory factors, inhibitory factors, and iRNAs and cDNAs, and their therapeutic applications in various disease conditions, would be the major directions in understanding how endogeneous stem cells response to these treatments.

There are several hundreds if not thousand of these factors available now for various types of experimentations and clinical testings, and even in human health and beauty applications.

I fully agree with MH Zhou on the large impact that determining the effects of growth and regulatory factors, siRNAs, overexpression of genes, etc may have on adult stem cells, leading to maintenance, expansion or even depletion of this population in vivo are one of the main issues, independently of the terminology changes described by Mohan Das Vaidya, although the new concepts and innovations he mentions could be relevant. Mohan, do you have any clue on what these could be?

Jose A.