A patient of Dr. Cheney's just told me that she will pay $12,000 for her next stem cell infusion in Panama. I don't know if Dr. Cheney has negotiated special rates with this company, or if this is the going rate.
Stanford researchers use DNA minicircles to induce pluripotency in stem cells from human fat
. 8 February 2010 00:06
Tiny circles of DNA are the key to a new and easier way to transform stem cells from human fat into induced pluripotent stem cells for use in regenerative medicine, say scientists at the Stanford University School of Medicine. Unlike other commonly used techniques, the method, which is based on standard molecular biology practices, does not use viruses to introduce genes into the cells or permanently alter a cell's genome.
It is the first example of reprogramming adult cells to pluripotency in this manner, and is hailed by the researchers as a major step toward the use of such cells in humans. They hope that the ease of the technique and its relative safety will smooth its way through the necessary FDA approval process.
"This technique is not only safer, it's relatively simple," said Stanford surgery professor Michael Longaker, MD, and co-author of the paper. "It will be a relatively straightforward process for labs around the world to begin using this technique. We are moving toward clinically applicable regenerative medicine."
The Stanford researchers used the so-called minicircles - rings of DNA about one-half the size of those usually used to reprogram cell - to induce pluripotency in stem cells from human fat. Pluripotent cells can then be induced to become many different specialized cell types. Although the researchers plan to first use these cells to better understand - and perhaps one day treat-human heart disease, induced pluripotent stem cells, or iPS cells, are a starting point for research on many human diseases.
"Imagine doing a fat or skin biopsy from a member of a family with heart problems, reprogramming the cells to pluripotency and then making cardiac cells to study in a laboratory dish," said cardiologist Joseph Wu, MD, PhD. "This would be much easier and less invasive than taking cell samples from a patient's heart." Wu is the senior author of the research, which will be published online Feb. 7 in Nature Methods. Research assistant Fangjun Jia, PhD is the lead author of the work.
Longaker is the deputy director of Stanford's Institute for Stem Cell Biology and Regenerative Medicine and director of children's surgical research at Lucile Packard Children's Hospital. Wu is an assistant professor of cardiology and of radiology, and a member of Stanford's Cardiovascular Institute. A third author, Mark Kay, MD, PhD, is the Dennis Farrey Family Professor in Pediatrics and professor of genetics.
The finding brings together disparate areas of Stanford research. Kay's laboratory invented the minicircles several years ago in a quest to develop suitable gene therapy techniques. At the same time, Longaker was discovering the unusual prevalence and developmental flexibility of stem cells from human fat. Meanwhile, Wu was searching for ways to create patient-specific cell lines to study some of the common, yet devastating, heart problems he was seeing in the clinic.
"About three years ago Mark gave a talk and I asked him if we could use minicircles for cardiac gene therapy," said Wu. "And then it clicked for me, that we should also be able to use them for non-viral reprogramming of adult cells."
The minicircle reprogramming vector works so well because it is made of only the four genes needed to reprogram the cells (plus a gene for a green fluorescent protein to track minicircle-containing cells). Unlike the larger, more commonly used DNA circles called plasmids, the minicircles contain no bacterial DNA, meaning that the cells containing the minicircles are less likely than plasmids to be perceived as foreign by the body. The expression of minicircle genes is also more robust, and the smaller size of the minicircles allows them to enter the cells more easily than the larger plasmids. Finally, because they don't replicate they are naturally lost as the cells divide, rather than hanging around to potentially muck up any subsequent therapeutic applications.
The researchers chose to test the reprogramming efficiency of the minicircles in stem cells from human fat because previous work in Wu and Longaker's lab has shown that the cells are numerous, easy to isolate and amenable to the iPS transformation, probably because of the naturally higher levels of expression of some reprogramming genes. They found that about 10.8 percent of the stem cells took up the minicircles and expressed the green fluorescent protein, or GFP, versus about 2.7 percent of cells treated with a more traditional DNA plasmid.
When the researchers isolated the GFP-expressing cells and grew them in a laboratory dish, they found that the minicircles were gradually lost over a period of four weeks. To be sure the cells got a good dose of the genes, they reapplied the minicircles at days four and six. After 14 to 16 days, they began to observe clusters of cells resembling embryonic stem cell colonies - some of which no longer expressed GFP.
They isolated these GFP-free clusters and found that they exhibited all of the hallmarks of induced pluripotent cells: they expressed embryonic stem cell genes, they had similar patterns of DNA methylation, they could become multiple types of cells and they could form tumors called teratomas when injected under the skin of laboratory mice. They also confirmed that the minicircles had truly been lost and had not integrated into the stem cells' DNA.
Altogether, the researchers were able to make 22 new iPS cell lines from adult human adipose stem cells and adult human fibroblasts. Although the overall reprogramming efficiency of the minicircle method is lower than that of methods using viral vectors to introduce the genes (about 0.005 percent vs. about 0.01-0.05 percent, respectively), it still surpasses that of using conventional bacterial-based plasmids. Furthermore, stem cells from fat, and, for that matter, fat itself, are so prevalent that a slight reduction in efficiency should be easily overcome.
"This is a great example of collaboration," said Longaker. "This discovery represents research from four different departments: pediatrics, surgery, cardiology and radiology. We were all doing our own things, and it wasn't until we focused on cross-applications of our research that we realized the potential."
"We knew minicircles worked better than plasmids for gene therapy," agreed Kay, "but it wasn't until I started talking to stem cell people like Joe and Mike that we started thinking of using minicircles for this purpose. Now it's kind of like 'why didn't we think of this sooner.
next month dr cheney sends another 13 to 14 patients to panama/costa rica for treatment stem cell.
dr from panama i last week ( i got lucky got him on the phone for free for 1 hr imagine that)says they had total of 60 patients in Costarica and panama not counting dr cheney and had great sucess rate..longest cfs who took stem cell now is 4 years no side effects so far only better..80% improved in all symptoms and his neurological improved 90%
Dr. Eric Gordon from GMA is also sending 20 or so CFS patients to Panama. Intriguing because GMA has collaborated early and often with WPI and Dr. Gordon is probably aware of Judy M saying it's NOT a good idea to get stem cells until the issue of possible XMRV reinfection of hematopoietic cells (stem cells develop into the full lineage of hematopoietic cells) is resolved.
Hopefully more of the patients that have gone will report here so we have all this info in one consolidated thread.
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The term 'autoimmune diseases' encompasses a spectrum of diseases whose clinical manifestations and, possibly, biological features vary widely. The results of conventional treatment are considered unsatisfactory in aggressive forms, with subsets of patients having short life expectancies. Relying on wide experimental evidence and more feeble clinical data, some research groups have used autologous hematopoietic stem cell transplantation (HSCT) in the most disabling autoimmune diseases with the aim of resetting the patient's immune system. Immunoablative conditioning regimens are preferred over their myeloablative counterparts, and some form of in vivo and/or ex vivo T-cell depletion is generally adopted. Despite 15 years' experience, published controlled clinical trials are still lacking, with the evidence so far available coming from pilot studies and registry surveys. In multiple sclerosis, clinical improvement, or at least lasting disease stabilization, can be achieved in the majority of the patients; nevertheless, the worst results are observed in patients with progressive disease, where no benefit can be expected from conventional therapy. Concerning rheumatologic diseases, wide experience has been acquired in systemic sclerosis, with long-term improvements in cutaneous disease being frequently reported, although visceral involvement remains unchanged at best. Autografting has proved to be barely effective in rheumatoid arthritis and quite toxic in juvenile idiopathic arthritis, whereas it leads to clinical remission and the reversal of visceral impairment in the majority of patients with systemic lupus erythematosus. A promising indication is Crohn's disease, in which long-term endoscopic remission is frequently observed. Growing experience with autologous HCST in autoimmune diseases has progressively reduced concerns about transplant-related mortality and secondary myelodysplasia/leukemia. Therefore, a sustained complete remission seems to be within the reach of autografting in some autoimmune diseases; in others, the indications, risks and benefits of autografting need to be better defined. Consequently, the search for new drugs should also be encouraged.
The prevalence of autoimmune diseases (ADs) in developed countries ranges between 2 and 7%, along with geographical differences and inclusion criteria. Namely, the term AD encompasses a rather wide spectrum of diseases, differing by clinical and possibly pathogenetic features.[5,7,8] A gross conventional distinction recognizes limited/organ-driven and systemic ADs, although a considerable degree of overlap exists between the two groups and some ADs do not fit in either.[47,9] In the former group, a 'peripheral' dysfunction is commonly held to play a major role, whereas in the latter, a 'central' immunological compromise is regarded as the pathogenetic clue.
Over the last few decades, investigation on experimental models has allowed a deeper insight to be thrown into the possible pathogenetic mechanisms of some ADs and has prompted the search for newer therapeutic approaches. The first step has been favored by the selection of AD-prone strains of laboratory animals, notably New Zealand Black (NZB) mice, who can develop systemic ADs bearing resemblance to systemic lupus erythematosus; in this setting, AD may be transferred to other mice strains through hematopoietic stem cell transplantation (HSCT) from NZB mice, whereas allogeneic HSCT from non-AD-prone mice strains is able to cure ADs in NZB mice AD-prone mice strains are considered the paradigm of spontaneous, systemic ADs and the possibility of transmitting or curing AD through allogeneic HSCT is regarded as a hallmark of stem cell disorder. In the opposite corner, other investigators have been able to induce in non-AD-prone mice AD varieties similar to their human counterparts, such as experimental allergic encephalitis (EAE) for multiple sclerosis (MS) and experimental adjuvant arthritis for rheumatoid arthritis (RA)[17,18] The same authors and other groups showed that experimentally induced ADs could be treated with syngeneic HSCT, suggesting an antigen-driven pathogenetic mechanism, In non-AD-prone mice strains, various experiments have shown that AD can be transferred from affected to healthy animals by syngeneic HSCT; conversely, syngeneic HSCT from healthy animals was able to cure some, but not all, AD varieties. Therefore, these results prompted two gross types of ADs in experimental animals to be distinguished: spontaneously occurring ADs, possibly deriving from a hematopoietic stem cell (HSC) disorder, which can therefore be cured through allogeneic but not syngeneic HSCT; and antigen-induced ADs, as experimental ADs, where autoreactive clones can be silenced by syngeneic HSCT.[17,2527] Although spontaneous ADs bear resemblance to human systemic ADs and experimental ADs to limited ones, it is unclear whether these models apply to human disease and, in an affirmative case, which pathogenetic model corresponds to each single human AD. Some milestones in the history of experimental ADs are summarized in Table 1 .
Anecdotal reports have shown that patients simultaneously suffering from a hematological disease and an AD could be cured, or at least achieve a durable remission, of their AD as well as their neoplastic disease by means of HSCT.[12,2830] These results were more commonly achieved after allogeneic HSCT, but similar cases have been reported also after autologous HSCT. Moreover, some AD varieties proved to be more liable than others to derive a benefit from HSCT and, also within the same disease, results were heterogeneous. On the other hand, ADs, both spontaneous and induced by priming agents such as cyclosporine and interferons, are fairly common complications after autologous HSCT, performed either for neoplastic or even ADs. However, post-HSCT, ADs are most often clinically self-limiting or are sometimes accounted for by laboratory findings devoid of a clinical counterpart. Worth mentioning is that organ-specific AD prevails among post-HSCT ADs, whereas the preponderance of patients undergoing autologous HSCT because of an AD are affected by systemic varieties of ADs. The lack of a common study design is a main explanation for the contradictory results mentioned to date, but, at the same time, the view is reinforced that AD varieties behave differently as to biology and response to treatment.
Studies on thymic lymphocytes after autologous HSCT have shown that, after a burst sustained by pretransplant memory cells, the organ is repopulated by likely harvest-derived naive T cells, and also the T-lymphocyte repertoire may significantly differ before and after autografting, thus suggesting the possibility of achieving an immune resetting through autologous HSCT, which in turn may exert a potential benefit on AD patients.
Finally, in a subset of AD patients with refractory disease life expectancy is so shortened to be comparable with that of oncohematological patients suitable for autografting.[46,47] In these patients, the dismal prognosis might reasonably counterbalance the risk of transplant-related mortality (TRM), at least in the setting of autologous HSCT. Nevertheless, long-term effects of autologous HSCT, notably secondary malignancies and metabolic syndrome, must be weighed in order to make a suited riskbenefit balance.
Relying on the aforementioned experimental data and on the rather feeble clinical evidence, different research groups set sights on finding out whether HSCT, either allogeneic or autologous, might be a suitable strategy to treat ADs.[4,5] Allogeneic HSCT may completely substitute the recipient's immune system with the donor's immune system and exert a graft-versus-autoimmunity effect, leading to cure of the AD. The diffuse application of this approach has been hindered by the fear of a high rate of TRM, which may result in a loss rather than a gain of survival in patients who, even with advanced disease, still have a median life expectancy of 3 years.[46,47] Moreover, graft-versus-host disease (GVHD) may induce clinical patterns mimicking ADs. As a consequence, autologous HSCT has found more extensive acceptance and interest. No different from autologous HSCT for neoplastic diseases, the original purpose was to destroy all autoreactive immune cells, and consequently to achieve 'cure' by inducing tolerance; finally, a less troublesome target was identified in its ability to counteract AD progression through a 'resetting' of the immune system.[30,49,5355]
I thought Dr. Cheney had also suspended getting more patients treated until more news about XMRV, but apparently he's sending another group down there in March (according to someone who just posted here), so that's interesting -- I wonder if his two recent conversations with Judy M. about XMRV changed his opinion? Does anyone know the answer to this?
As I understand it, stem cells would typically be screened for the pathogens that the Red Cross would screen for, so theoretically if the CDC is actually worried about the blood supply and XMRV, screening for XMRV will occur as soon as there's some consensus on the right screening labs, since the Red Cross already includes other retrovirus/infectious disease screening for the national blood supply.
There was a case last year in the medical literature of someone with HIV/AIDS seroconverting to HIV-negative after receiving stem cells because he received a stem cell transplant from a donor with natural HIV resistance, so I don't think it's possible for stem cells to eradicate the XMRV retrovirus IF someone was lucky enough to get the right donor: otherwise it would probably just be the improvements in immune parameters that would give one a fighting chance against XMRV, since obviously there are people in the population carrying XMRV who are not sick with ME/CFS so it's possible to keep it suppressed somehow, though nobody yet knows how or for how long.
Hello everyone, I had a few questions for Molly regarding preparation for receiving the CS infusion.
1) Aside from attending Cheney's lecture, were there any other resources you found particularly helpful in learning about the CS infusion process?
2) You mentioned earlier that you spent 90 days working on your gut. Would you mind giving a brief overview of your protocol?
3) Finally - one of the worst problems I (and probably most others) have with this disease is the inability to do any intense exercise without crashing for weeks. Are you currently able to do hard exercise without fearing a long crash?
Thank you so much for sharing your story. I think you have helped a lot of people by doing this and I hope you continue to improve.
I saw my Lyme doc last week and he brought up Cheney's use of stem cells, said he/Cheney has been having good success with them, and then he mentioned stem cells they can make from yourself. Autologous stem cells, I think? He thought I should consider doing this when I'm done addressing some of my current issues (bartonella and biofilms) in order to help regenerate my body. It'll be awhile before I could get to doing this but I thought I'd start researching now. I'm a bit confused because my Lyme doc gave a talk last year at a Lyme conference where it sounded like he was not recommending stem cells due to the risk of cancer. I assume autologous stem cells would not have that risk? Can anyone point me to where I might research more about this?
The progress you've made since your stem cell treatment sounds like a minor miracle.
It's great to hear that your cognition, stamina and ability to bounceback are all improving.
And thanks for reporting your experience so extensively and answering so many questions. Stamina, right there.
A huge help to those of us just beginning our own research.
I also have had high EBV, CMV and HHV-6 titers as well as high RNAsel.
Hope today you're doing something more fun than fielding queries so I won't inundate you with a million questions now.
(read: I would love to ask a million questions about your treatment). Like Dhearns, I'd love to know what other
resources aside from the Cheney lecture helped in your research, and also curious about your 90 day gut protocol prior to treatment.
I may try to gather up a group of folks that are very interested in learning more and perhaps
we could pool our questions and save you some time.