√ 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.

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√ Possible Safety Concerns From New Stem Cell, Rpe Vision Loss Study Report

With stem cells for vision loss, first we want to be sure a treatment won’t make things worse.

Several teams around the globe are rigorously studying stem cell-based approaches to vision loss via regulatory-compliant studies including for macular degeneration with some results cautiously upbeat on safety from early phase analyses, but data from a new study on the use of human embryonic stem cell (hESC)-derived retinal pigmented epithelial cells (RPEs) for macular degeneration are concerning in some ways.

Figure 1, Mehat, et al. AAO Journal 2018

The paper in the AAO Journal from a team led by James W.B. Bainbridge at UCL is entitled “Transplantation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells in Macular Degeneration.”

The data here suggest that this kind of approach has some potential issues. While every stem cell study is unique, this paper also has important implications for the other studies out there using either hESC or human induced pluripotent stem cells (hIPSC) to make RPE for various forms of macular degeneration.

First, the bit of encouraging news was, “We found no evidence of uncontrolled proliferation or inflammatory responses,” which is something. You can see fundus images of the patients’ retinas in Figure 1.

However, they did not find a treatment-related improvement in vision so efficacy is not really supported so far with the analysis of the 12 patients for 13 weeks. Longer-term improvements are possible, but relatively less likely I think.

Also, there was a potential adverse event in a patient receiving the highest dose of RPEs. The authors wrote, “In one instance at the highest dose, localized retinal thinning and reduced sensitivity in the area of hyperpigmentation suggested the potential for harm.”

You can see supplemental Figure 3 below and note the drop in pigmented thickness in patient 10, especially as time went on in the study.

Supplemental Figure 3, Mehat, et al. AAO Journal 2018

What does this mean? It sounds like the RPE at the higher dose may have actually made the retina less healthy and impaired vision, which is worrisome, but more data are needed to be sure.

Other events are described that potentially could be concerning too including some possible immune rejection in 2 patients and ectopic RPEs in a number of patient, although the latter apparently didn’t clearly affect function:

“A reduction in the pigmentation density at the site of transplantation, evident in 2 participants (at month 6 in patient 9 and at month 12 in patient 4) may reflect rejection of pigmented donor hESC-derived RPE cells, although no associated change in retinal function was apparent.”

and

“In our study, intraocular administration of carefully obtained hESC-derived RPE cells resulted in the development of pigmented foci in the vitreous cavity in 2 participants and on the surface of the inner retina in 2 participants, suggesting reflux of donor hESC-derived RPE cells from the subretinal compartment into the vitreous cavity. Despite the presence of preretinal or intravitreal pigmentation, no associated adverse effect was evident.”

I asked Dr. Bainbridge for his perspectives on these points and the paper overall, and he had these comments:

“The hyperpigmentation is consistent with the presence of donor cell material but we have no evidence that the cells are surviving.

We are not able to biopsy the engrafted area safely to determine donor cell survival with confidence.

We were not able to detect any improvement in retinal function in the hyperpigmented areas despite detailed perimetric analysis.

Further analysis will determine whether the intervention can protect sight in the longer term.

Localised retinal thinning and reduced function may be a consequence of the natural history or the intervention.

Detailed topographical analysis of structure and function is needed to determine the safety and potential efficacy of such interventions.”

The paper ends cautiously this way:

“The evidence of safety broadly supports the rationale for further studies to explore the impact of intervention at an earlier stage of degeneration when surviving photoreceptors cells may stand to benefit with improved function and survival. However, instances of focally reduced sensitivity and thinning in hyperpigmented retina at higher doses of hESC-derived RPE cells suggest the potential for harm and indicate that intervention at earlier stages of degeneration should be approached with caution.”

Although other similar studies have reported some adverse events (e.g. see here), they seemed mostly likely to be procedural rather than cell-related. The outcomes here warrant more thought and some extra caution moving forward.

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√ New Stem Cell Pubs Including Artificial Human Embryo Work

In this post I list some recent interesting stem cell and science pubs including artificial human embryo research.

Artificial human embryos. Screenshot of Fig 4d Nat Cell Bio 2019 Simunovic et al. From the caption, “Molecular signature of EMT: downregulation of the adherent junction protein E-CAD and the expression of N-CAD in the BRA+ region. Images are representative of three imaged 3D colonies stained with the BRA, E-CAD and N-CAD combination.”

Engineered human embryo research continues.

Scientists for years have been advancing the types of embryo-like structures made from both human and other creature’s cells. In a new Nature Cell Bio pub entitled, “A 3D model of a human epiblast reveals BMP4-driven symmetry breaking”, a team led by Eric Siggia pushed this further. See an image from the paper I’ve included that shows signs of the primitive streak and epithelial to mesenchymal transition (EMT) in the embryo-like structures.

They report being able to model a human epiblast of a sort from human embryonic stem cells. From the abstract, “Here, we use human embryonic stem cells to generate an in vitro three-dimensional model of a human epiblast whose size, cell polarity and gene expression are similar to a day 10 human epiblast. A defined dose of BMP4 spontaneously breaks axial symmetry, and induces markers of the primitive streak and epithelial-to-mesenchymal transition. We show that WNT signalling and its inhibitor DKK1 play key roles in this process downstream of BMP4.”

You can read a couple past posts I did on pubs about artificial embryos here and here.

You can imagine that engineered human embryo research raises ethical issues too. In an NPR piece by Rob Stein, he has this quote on that level:

“It’s very exciting work,” says Insoo Hyun, a bioethicist at the Case Western Reserve University and Harvard Medical School who was not involved in the research. “But it does send folks down the road to thinking very seriously about where the limits may be ethically for this work.”

What do you think of engineering human embryo-like structures?

Here are some other interesting recent pubs and news.

• Direct Reprogramming of Human Neurons Identifies MARCKSL1 as a Pathogenic Mediator of Valproic Acid-Induced Teratogenicity


• Stress-Induced Changes in Bone Marrow Stromal Cell Populations Revealed through Single-Cell Protein Expression Mapping


• A new paper totally hyped in the media related to stem cells for baldness: Tissue engineering of human hair follicles using a biomimetic developmental approach.


• Not stem cell related, but very cool work covered in New York Times by Carl Zimmer, “New Weapons Against Cancer: Millions of Bacteria Programmed to Kill.”


• Taking ‘baby steps’ to human organs in livestock

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