√ Stem Cell Transplants: Hipsc And Hesc Behave Similarly In Brain Often Fuse With Host Cells

What happens following pluripotent stem cell transplants into the brain? Are human IPS and ES cells going to function similarly in this context?

We recently published a new translational paper on the behavior of human pluripotent stem cells when transplanted into the 4dukt mouse brain in collaboration with my great UC Davis colleague, Dr. Veronica Martinez-Cerdeno.

Martinez-Cerdeno Figure 6

Martinez-Cerdeno V, Barrilleaux B, McDonough A, Ariza J, Yuen B, Somanath P, Le C, Steward C, Horton K, Knoepfler P. Behavior of xeno-transplanted undifferentiated human induced pluripotent stem cells is impacted by microenvironment without evidence of tumors. Stem Cells Dev. 2017 Jul 10. doi: 10.1089/scd.2017.0059.

We are excited about this paper for a number of reasons. One of the things that makes it pretty unique is that we in parallel transplanted undifferentiated human iPSCs and human ESCs (hIPSC, hESC), whereas most studies pre-differentiate pluripotent stem cells. Even so we did not observe teratomas in the transplant recipients’ brains, which surprised us. It was also unexpected that we found that the mice did not strongly reject the transplants despite the animals being immunocompetent. The end result is that the mice survived without tumors and we got reasonable engraftment of both cell types and could compare their behavior.

You can see Figure 6 of our paper below, showing the fate of transplanted hIPSC where green staining highlights human cells and red staining is for the different indicated fate markers. Most human stem cells took on the appearance of NeuN+ neurons, while in contrast we did not observe human astrocytes. The numbers of human cells (and their fates) present in transplant recipient mice varied by region of the CNS although they were all injected stereotactically in the same location in the lateral ventricle, suggesting that microenvironment impacts transplanted cell behavior in key ways.

Why no tumors? In theory, immune rejection could have explained the lack of tumors. The mouse immune system perhaps killed the specific subpopulation of cells within the injected pluripotent stem cell cultures that had tumorigenic potential. It is also possible and I think more probable, since we observed a surprisingly high rate of fusion of the transplanted hIPSC or hESCs with host mouse brain cells, that this fusion eliminated tumorigenic potential.

We theorize that after human cell fusion with mouse cells, that nuclear fusion quickly followed since we did not observe multi-nucleated cells. The cell fusion we report is something that everyone studying cell transplants should look for in their studies. In theory transplanted stem cell fusion may not be entirely a bad thing as it could be exploited for drug delivery if we can better understand the mechanisms of fusion, but fusion can also potentially complicate apparent findings, especially if it isn’t on your radar screen.

Just because we didn’t observe tumors, doesn’t mean that undifferentiated human pluripotent stem cells would be safe to use clinically (I don’t think they would be) and other groups have reported teratoma in similar although not identical translational studies. In addition, we found in contrast to our human cell results that transplantation of mESCs did lead to teratoma or similar kinds of tumors such as one that appeared like a teratocarcinoma. It remains unclear why the mouse cells formed tumors while the human did not but xenotransplantation may lead to other effects such as the aforementioned cell fusion that impact biological outcomes.

There are a few limitations to our study on pluripotent stem cell transplants. For instance, it isn’t clear how a wider panel of hIPSC and hESC would behave, and some may be more predisposed to form teratoma. Also, we don’t know if the xeno nature of our transplants catalyzed the fusion and that leaves it unclear how much human stem cells would fuse with host differentiated cells following transplants. The clearest indicator of the rate of engraftment was by staining with a human-specific antibody and we also got some nice data by FISH, but while we tried a quantitative approach using qPCR to more precisely measure human gDNA present in the transplanted murine brain, this assay only worked in certain conditions. Most likely the qPCR assay sensitivity was limited by various factors including using fixed brain tissue DNA as starting material.

Overall, hIPSC and hESC behavior was quite similar in our study, further supporting the growing notion that reprogramming produces human cells generally equivalent to hESC, but a unique thing here in our study is that we found this similarity even within the transplant setting context in the brain (not just in a dish).

Many interesting follow ups could build on this work’s foundation including examining the impact of brain injury on transplanted undifferentiated hIPSC and/or hESC behavior. I’d be interested in any thoughts you have regarding the data in our paper on pluripotent stem cell transplants.

Sumber aciknadzirah.blogspot.com

√ Adverse Event In Ips Cell (Ips細胞) Trial For Vision Loss In Japan: Initial Perspectives

Mandai, et al. NEJM 2017 Figure 1C

A team of researchers at RIKEN and Kobe City Medical Center reported at a press conference today a serious adverse event in a clinical trial participant receiving an induced pluripotent stem cell (IPS cell)-based therapy for vision loss.

According to Japan Times and Yahoo! Japan (in Japanese), the patient in question in the IPS cell trial for macular degeneration developed a persoalan with his eye following the cell transplant. From Yahoo! Japan:

“The patient had an operation on Monday to remove pre-retinal membrane, deemed to be the cause of retinal edema, as the team was unable to improve the symptoms despite the administration of a steroid and anti-vascular endothelial growth factor medication.”

You can see an example of an experimental IPS cell-based transplant of RPEs in the team’s important paper from last year above.

The team leader, Dr. Masayo Takahashi indicated in a press conference that:

“We cannot deny the causal correlation with iPS cells,” Masayo Takahashi, a researcher at Riken who heads the team, said at a news conference.

Takahashi said the symptom “falls into the category of serious cases,” as it requires hospital admission for treatment, but that it is “neither a matter of great urgency nor life-threatening.”

I’m not clear if the IPS cell-derived retinal pigmented epithelial (RPE) cells grew or assembled into this “pre-retinal membrane” or it was more related to the injection protocol itself, but the words “causal correlation with iPS cells” suggests they played a role. Update: However, a translation of the Japanese article now suggests the opposite, that perhaps the cells themselves were not an issue:

“Riken project leader, Masayo Takahashi told that she considers the cause of adverse event the surgical method used for cell transplantation, not iPSC itself.”

It’s reassuring that Dr. Takahashi also said the incident should not interfere with the future of the trial:

“She said the incident should not affect the future process of the clinical study of transplantations of iPS-derived retinal cells in patients with severe eye diseases.”

I’m trying to better understand what happened to cause the problem, but need more information to have a better sense of what the implications might be. A quick Internet search of “pre-retinal membrane” suggests it can be due to scarring of the outer surface of the retina.

Notably, Japan’s regenerative medicine law mandates reporting of such adverse events:

“The team made the announcement in accordance with the law requiring medical institutions to report to the Minister of Health, Labor and Welfare a patient’s illness or worsening condition suspected to have been caused by regenerative medical techniques.”

In the past, a different iteration of the IPS cells for vision loss study in Japan was halted reportedly due to regulatory changes and potentially because a patient’s IPS cells had some mutations. Japan’s IPS cell efforts have switched gears in recent years to a more allogeneic (stem cells from other people rather than from yourself, which is called autologous) focus.

As there are more and more stem cell trials of various kinds, adverse events are to be expected, should be reported as this one was, and with proper controls can be understood in terms of risks as products navigate the process.

This all goes to show that stem cell-based clinical trials are more of a non-linear marathon than a sprint from points A to B, whether the clonal work is based on 4dukt, embryonic or IPS cells.

If I hear from Dr. Takahashi on more specifics of what happened and why or get more information another way I’ll update this post or do a new one to follow up.

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√ Crispr To Make Ips Cells: Review Of New Ding Lab Paper

CRISPR and IPS cells are two of my favorite things so when they come together as in a cool, new paper from Sheng Ding’s lab, I’m excited to read it and curious as to what the scoop is.

Since 2006-2007 when mouse and human reprogramming were first reported, many different methods have been explored to make induced pluripotent stem (IPS) cells. Along the way we’ve all learned quite a lot about how pluripotency is regulated, whether it is in the context of maintenance or induction of this unique stem cell state.

It was only a couple of years ago that all-chemical approaches to making IPS cells via reprogramming were reported (e.g. here). In the early years it seemed like every new reprogramming method could be a high-profile paper. Then methodological advances were eventually seen as not quite so big a deal. Now we have this one that strikes a chord in some ways.

This week’s Ding lab paper is Liu, et al. and is entitled, “CRISPR-Based Chromatin Remodeling of the Endogenous Oct4 or Sox2 Locus Enables Reprogramming to Pluripotency.” You can see the graphical abstract below that summarizes the work visually.

Liu et al Cell Stem Cell IPS cells 2018

The main gist of this paper is that CRISPR epigenetic (not gene editing) systems, which more broadly have previously been shown to have utility in activation or repression of genes through tethering transcriptional regulatory domains to dCas9, can be used to turn on two important pluripotency genes, Sox2 and Oct4, in mice and lead to formation of IPS cells. Notably, gene activation via CRISPR (termed CRISPRa) has been shown previously to turn on pluripotency genes (e.g. here), but the authors say not for full reprogramming to make IPS cells before.

There are a number of interesting things about this paper too. The fact that CRISPRa of Sox2 was reportedly sufficient to lead to some bona fide IPS cell formation was somewhat surprising since this gene is not strictly pluripotency-specific and to my knowledge it has not been previously shown to lead to cellular reprogramming on its own. The authors did note though that only 0.1% reprogramming efficiency was achieved with activation of Sox2 even though 20% of cells turned this gene on with CRISPRa, which suggests most of the time other things need to happen too, or as the authors put it, “that sole activation of endogenous Sox2 is not determinant to pluripotent cell fate in our CRISPRa context.” Still, they report that turning on Sox2 is enough to get the ball rolling successfully some of the time. If this is reproducible, then in non-pluripotent cells that are Sox2+ such as certain neural precursors, there must be mechanisms to inhibit progression to a pluripotent state such as repression of Oct4.

CRISPRa of just Oct4 also did the trick to make IPS cells, which is still somewhat surprising since one factor reprogramming using other methods hasn’t been reported that often that I know of at least using other delivery methods. Specific epigenetic targeting of both the Oct4 enhancer and promoter worked a lot better. Interestingly, it seems that the Oct4 promoter provides a quick burst of expression and the enhancer may provide lower magnitude, but more persistent activation.

In the context of this study’s CRISPRa system, histone acetylation was also shown to play an important role in activation to promote reprogramming to an IPS cell state via swapping in p300 into the SunTag system.  However, it’s not clear if other forms of epigenetic activation such as perhaps via induced H3K4me3 (a mark that sharply marks the TSS in promoters of active genes) could yield similar results, perhaps even with relatively lower acetylation levels. There are still many interesting questions here overall.

This study was also focused on mouse cells only so another important thing to try will be CRISPRa production of human IPS cells. Hopefully that’ll work too. One other limitation I saw in the paper was that it didn’t do an unbiased search for off-target transcriptional effects of CRISPRa, but just looked at a finite set of predicted transcriptional off-targets.

This kind of system may also work for direct reprogramming of one cell type to another such as production of neural precursor cells from fibroblasts, a process sometimes referred to as transdifferentiation. It’s possible in this paper that they successfully got production of IPS cells whereas others didn’t because here they were able to induce much higher levels of expression including via the SunTag system. Addition of small molecules to the media gave things a boost as well.

Overall, I liked this paper for its fairly novel technical approach, its potential to open the door to a new method of non-genetic reprogramming, and insights into pluripotency.

Sumber aciknadzirah.blogspot.com

√ Perspectives On High-Profile Stem Cell Retraction In The Making From Kyoto University

Screenshot from NHK video on IPS cell paper problems

A researcher working at Kyoto University has reportedly engaged in misconduct leading to an institutional request for retraction of a paper from the journal Stem Cell Reports.

The paper in question appears to have rather large-scale problems:

“Kohei Yamamizu, a specially appointed assistant professor at the university’s Center for iPS Cell Research and Application, falsified all six main figures in the paper that claimed he had succeeded in generating a brain structure in vitro using iPS cells, according to Kyoto University.”

The Center for iPS Cell Research and Application, also known as CiRA, is a flagship induced pluripotent stem (IPS) cell institute and is run by IPS cell discoverer and Nobel Laureate Shinya Yamanaka.

Yamanaka, not involved in the research in question, nonetheless was quoted about this stem cell retraction and understandably down about it. I would be too in that kind of situation. I don’t see that Yamanaka could have done anything to prevent this though so it’s clearly not his fault in any way:

“I am very regretful for not being able to prevent the dishonesty,” said Yamanaka, professor at Kyoto University who won the Nobel Prize in medicine in 2012 for discovering induced pluripotent stem cells, or iPS cells.

“I will take (the incident) seriously and provide education necessary to prevent similar misconduct from happening again,” Yamanaka told a press conference.”

The paper in question seems to be a 2017 article entitled, “In Vitro Modeling of Blood-Brain Barrier with Human iPSC-Derived Endothelial Cells, Pericytes, Neurons, and Astrocytes via Notch Signaling” and the alleged misconduct was from first author Kohei Yamamizu. The PI on the paper is Jun K. Yamashita. I noticed that another recent stem cell paper on which Yamamizu and Yamashita are authors has a correction.

It remains unclear how the alleged misconduct was detected. A newer NHK report (apparently this story also made the nightly news in Japan; see screenshot above from news video) also weighs in and indicates some data was fabricated:

“The university set up a panel to investigate the credibility of the paper after doubts were raised about it.

The investigation found 11 images were fabricated or altered to back up the paper’s findings.

The university determined that Yamamizu, who had analyzed the data and made the illustrations, was responsible for the wrongdoing.

He has reportedly admitted to the falsifications, saying that he wanted to make the paper more visually appealing.”

Even an ugly or relatively plain-looking paper is better than a flashy one if the ugly one is more accurate and the data rock solid. Sometimes I think certain reviewers unrealistically expect biology to be so simple as to produce perfect looking data and figures. Biology is most often not that straightforward. This is of course no excuse for misconduct.

The stem cell field has had quite a few retractions over the years, but I was thinking that in 2017 there hadn’t been many high-profile ones compared to past years. At one point I compared stem cell retractions to those in the cancer field (which do you think had more?) Perhaps the most well known stem cell retractions of all are those of the two Nature papers on STAP cells, which were critically reviewed early on here at The Niche.

We’ll see what fate awaits this paper and how this unfortunate situation plays out. I don’t see it as a setback for the IPS cell field at all, but still should be taken seriously for what it is.

Sumber aciknadzirah.blogspot.com

√ Tgif: Guns, Stem Cells, Science Weekends Reads Good News

I’ve got some TGIF recommend reads on stem cells, etc, but first, what a week, right?

Wakabayashi, et al. Cell Stem Cell Figure 1F

We need more action on preventing gun violence. It’s a uniquely American health crisis that doesn’t have to be. There’s no real, common sense reason that gun control can’t reduce mass killings of our kids and others like the latest one in the Florida high school. That kind of gun control won’t interfere with hunting or other legitimate gun ownership. There are clearly going to be politicians who will not cooperate with even sensible gun control and we should vote them out of office as soon as possible. Okay, I had to get that out there. More gun research would help too.

Back to just science.

Here are some cool science and stem cell recommended weekend reads, headlines, and some good news out there.

“Induced pluripotent stem cells could serve as cancer vaccine” and the source paper led by Joe Wu, which used IPS cell technology for translational cancer research. Very cool. This is at the top of my reading list for the weekend.

“Adult endothelial stem cells can make fully functional blood vessels” and the source paper here also at Cell Stem Cell. Also, see cool image from the paper showing generated blood vessels.

“Will 100 be the new 60? Stem cell start-up that raised $250 million could extend lifespan by decades, help cure cancer”. Hype award for the week and maybe the year (not just this media piece specifically, but this biotech story on Celularity more generally, which I’m going to post more about next week).

“Stem cells zapped with radiation can protect mice from cancer”

Grandmother loses life-savings for stem cell ‘cure’ at St. Louis clinic. Many disturbing elements to this story, but it is all too common.

Opinion piece in Cell Stem Cell: “Could Failure in Preimplantation Genetic Diagnosis Justify Editing the Human Embryo Genome?” My answer would be “No”.

“Temporal Layering of Signaling Effectors Drives Chromatin Remodeling during Hair Follicle Stem Cell Lineage Progression” from a team led by the great Elaine Fuchs, of course.

Good news for CIRM and California, as our stem cell agency receives 1st royalty check, a small one but still a big milestone.

Good news on the prostate cancer front with new drugs effective at delaying detectable metastatic spread (for those of you who didn’t know, I am a prostate cancer survivor, now 8+ years out).

Sumber aciknadzirah.blogspot.com

√ Japan Conditionally Approves New Ips Cell-Based Heart Study

Where do things stand with IPS cell translational research?

The newest development is that regulators in Japan just have given conditional approval to an Osaka University team to an induced pluripotent stem (IPS) cell-based study for ischemic heart disease.

Screenshot of Yoshiki Sawa slide on IPS cell approach for heart failure, which is similar to approach for ischemic heart disease.

For years IPS cell-based products have been on-again off-again in active clinical study for macular degeneration, work led by Dr. Masayo Takahashi. The new heart disease study is led by Yoshiki Sawa. This appears to be the third conditionally approved IPS cell study in Japan. I’m not clear on whether both other studies included in the total of 3 here are from Dr. Takahashi or not.

Here is information on the study design:

“The approved plan will cover patients with ischemic cardiomyopathy, or hearts weakened by narrowed or blocked arteries preventing blood from reaching parts of the heart muscle. Three people aged between 18 and 79 will have two round cell sheets made from the iPS cells of other people attached to the surface of their hearts. The sheets will each be 0.05 millimeters thick and several centimeters in diameter. Researchers will examine the safety of the procedure, checking for cancer, immunorejection and other such signs, and will also observe changes in the function of the patients’ hearts.”

It has a number of key differences with the previous conditionally approved studies beyond the organ of focus. Perhaps the most striking aspect of the new study is that it will use far more cells:

“This is the third case of clinical research involving transplants using iPS cells to be approved. In the previous two cases, groups including the Riken research institute are transplanting several hundred thousand cells in the treatment of intractable diseases. The number of cells in the latest research is far greater — at around 100 million. As there is a risk of iPS cells turning cancerous, it will be crucial for researchers to guarantee the safety of the process.”

Ideally, we don’t want any undifferentiated IPS cells in the transplanted product, but that’s a tall task with 100 million cells. Past studies have suggested that not all IPS cells have tumorigenic potential including in a transplantation context so a few amounts 100 million overall may not cause problems. This will have to be carefully monitored as stressed in the  Mainichi piece. From a second Mainichi article we have a quote on this issue from Dr. Sawa:

“Even if a tumor does develop, because the cells come from another person, it is expected that it will disappear after the suppressants are withdrawn and the person’s natural immune system kicks in.”

I hope he’s right about that.

This is exciting investigational work, but high-risk in my view. It will be interesting to follow.

You can learn more from a slide set from Dr. Sawa’s research here (and see image above from one such slide).

Sumber aciknadzirah.blogspot.com

√ Japan Stem Cell Oversight System Too Lax?

The HeartSheet product shown here is advancing in the pipeline rapidly in part due to the Japan system of conditional approval of stem cells

The Japan stem cell and regenerative medicine oversight system has been designed to move investigational therapies forward fast, much faster than the systems in other countries such as the U.S. But how well is it working so far?

It’s an important question both for that country and more globally as the Japanese system was touted by some as a model and a reason why the U.S., to remain more competitive in this industry. Some of the same folks felt that the U.S. should also adopt a conditional approval approach and lobbied in past years for the REGROW Act. I felt REGROW was an extremely risky piece of legislation, especially in its initial incarnations. The regenerative medicine provisions in the 21st Century Cures Act, which became law, were more measured and balanced. We’ll see how it turns out now that it is being implemented in a practical sense in the real world. In the mean time, some of us have continuing concerns that the oversight system for stem cells and regenerative medicine in Japan may be too permissive.

Within the Japanese regenerative medicine oversight system, clinical studies involving induced pluripotent stem (IPS) cells such as the one I blogged about last week for heart disease involving the so-called HeartSheet led by Yoshiki Sawa can take steps forward with less pre-clinical and/or early clinical trial data than one might expect. The news about the heart IPS cell study has generated both buzz and some criticism that it is too risky.

The critics include Nature, which in an editorial this week called for better oversight in Japan and more preliminary data before stem cell therapies are sold to patients. The editorial was subtitled strongly, “Japan needs to demonstrate that a promising therapy for damaged hearts works as claimed.” Also, the editors wrote (emphasis mine), “The government needs to move quickly to make sure that evaluation of the HeartSheet therapy is as rigorous as promised. As more treatments emerge, officials should make sure that — fast track or not — they have a valid claim to efficacy before being sold to patients.”

A NatureNews piece by David Cyranoski also highlighted some concerns about this situation:

“Yoshiki Yui, a cardiologist at Japan’s Kyoto University, says that, as well as meeting the requirements for safety, researchers should show that their treatment is effective, which would require testing it in larger numbers of people than are currently required. The evaluation process should also use randomized, controlled clinical trials, the gold standard for demonstrating efficacy in medical research, he says.

The iPS-cell therapy has potential, Yui adds, but under the current approval system, “we won’t know if it works or not” because it won’t have been tested in a controlled trial. “The biggest masalah is there’s no adequate system of evaluation in Japan,” says Yui.”

The Japanese government contends the current system is fine. You can see more about Yui’s concerns including from a medical science level in a piece here.

It seems to me that it is only logical that stronger proof of efficacy including data from rigorously designed studies with proper control subjects is needed at the pre-market step. Less data equals higher risk for patients even if it also means speedier clinical science progression, but the question is where is the sweet “speed” spot of risk versus potential benefit? My own view is that Japan is still somewhat leaning too far to the speedy side at the moment. That could change.

Meanwhile, stem cell oversight is evolving in interesting ways elsewhere across the globe too. For instance, here in the U.S. we have the regenerative medicine advanced therapy (RMAT) designation program, which is also a regulatory experiment in accelerating stem cell oversight but does not allow selling incompletely tested stem cell therapies to patients, and also this week we have the new Right-To-Try law that may impact the stem cell field potentially interfering with some elements of oversight.

More will be coming on Right-To-Try and stem cells in future posts.

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