K. John McLaughlin: So I'm from Children's Hospital, Nationwide Children's Hospital, and I've been there three years. And before that, I was at Penn Vet, I taught in the vet school there. And before that, I was in Germany at the Max Planck in Freiburg, and I've been doing mainly developmental biology and stem cell research, particularly for the last 13 years. And I currently have a joint role. I have the directorship of the Transgenic Mouse Core at Children's Hospital. So I have the staff from the core lab and my lab, which is two postdocs and two technicians, actually three technicians total. And we have the rotation students coming through, no graduate students at the moment. So there's plenty of slots available and plenty of postdocs to help supervise the graduate students. So we've had quite a few rotation students come through from this program, but nobody that's stuck so far. So I just thought I'd give you a quick ten-minute summary, which is the request by Geoff, and I'll keep it to ten minutes. So, I got into stem cells because I was working in developmental biology, and about 13 years ago, there was this big controversy about the use of human embryonic stem cells for research, and the prospects that this slide here tends to exaggerate. [Image of The New York Times Magazine Cover, titled, "Racing Toward Immortality" featuring a man and woman on scooters] So the holy grail was to produce autologous stem cells, human embryonic stem cells that could be differentiated into any tissue. So it had to be autologous. So cloning was obviously the most attractive way of doing that because this was in the wake of Dolly and the cloning of mice embryos back around 2000. So, this paper came out in 2005, and it was retracted the next year because it was found out that the embryo, that was the single line that was actually a reality, was actually derived from a parthenogenetic embryo. [Text on screen Patient-Specific Embryonic Stem Cells Derived from Human SCNT Blastocytes. The text also lists the paper's 26 authors and citation.] So they didn't actually get a fertilized egg at all, so that was unfortunate. But actually, just very recently, so just last month, a group from Portland, who's been working with non-human primates have actually cloned human embryos. He's cloned human cells, adult somatic cells, and generated embryonic stem cells, which have proven to be valid or viable as much as they can determine. So this has really opened the possibilities for autologous stem cell transplants. [Text on screen Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer. The text also lists the paper's various authors and citation.] So the options for getting autologous stem cells, embryonic-like stem cells, is three different types. So there's fertilized embryos, which is what the controversy was about around 2000 with the Bush administration. And they were just using surplus embryos from human IVF to generate ES cells, which is great because there's a huge supply of embryos that are not used. But the problem is, none of them are autologous, so they're kind of defunct. So the options for autologous stem cells are cloning or somatic cell nuclear transfer. And the more recent approach of induced pluripotent stem cells, where they throw a bunch of factors on basically any adult cell type and turn them back to embryonic stem cells. [Text on screen Sources and therapeutic application of pluripotent stem cells] [Image of fertilized embryo cell, SCNT, somatic cells: IPS, with and arrow pointing to pluripotent embryonic stem cells with an arrow branching into 3 with different cells ultmatelt pointing towards a diagram of a human] So, either three of these methods will give you embryonic stem cells or embryonic-like stem cells, and these can be differentiated into various tissues, which can be transplanted. So my angle in this story, hang on, yeah, so IPS cells had a few problems because people thought that they originally were abnormal, but the technology has improved now, and they've examined them compared to true embryonic stem cells, and they found that actually, the defects they see in these induced pluripotent stem cells from adult somatic cells are pretty much the same as those from true embryonic stem cells. So the technology is developing. So I think that eventually, IPS cells, which we patient-derived, are going to be the optimal source. [Text on screen Genetic aberrations in human IPS cells are similar to embryonic stem cells] [Graph on screen demonstrates the relationship between recurrent abnormalities and percentage of abnormal cultures] So my angle on this was to look at parthenogenetic cells. So, parthenogenesis is when you get development without the contribution of the male genome. And this happens, or it happens in nature by activation of development, either by retention of the second polar body or by other mechanisms, depending on the species, and there's several animals around that actually exist solely by parthenogenetic reproduction, such as the Amazon molly. And there's also examples of a turkey that was developed by USDA that was actually supposed to be producing derived pathogenesis. And there was this Komodo dragon in the London Zoo, a female that was alone for several years and suddenly produced 100 male lizards without any reproduction. [Text on screen Uniparental Mouse Embryos Do Not Develop] [Image on screen of the Amazon molly, a turkey, and a komodo dragon. A diagram of a MII oocyte activating into pathogenic (PG).] So it's quite interesting, but the problem is, in mammals, it doesn't work. So obviously, you can't have parthenogenesis in humans, otherwise, us men would be superfluous. [Image changes to a representation of Mill oocyte becoming parthenogenetic, with an arrow stating that this leads to poor development of embryonic tissues. Image of turkey remains on screen] So what happens? So if you have a parthenogenetic development, where you have two maternal genomes, they developed for four to nine days, so they developed early somite stages in some circumstances. So, if you use tricks in the lab where you do nuclear transplantation, you can actually remove the female pronucleus just after fertilization and put in another male pronucleus. Which has come from the sperm and get two paternal genomes, and you get these, what are called androgenetic embryos, and these only develop for three to eight days. [Image illustrating the development of uniparental embryos: parthenogenetic, gynogenetic, and androgenetic] So there's obviously something that's stopping development, and that's called genomic imprinting. So if you have two, there's a core epigenetic difference between the maternal and paternal genome, which precludes viable development, and that is due to the fact that there's genes which you're only expressed from your maternal or paternal allele. So IGF2, for example, is only expressed from the allele you inherited from your father. H19 is only expressed from the allele you inherit from your mother. So if you have two paternal two maternal copies, for some genes, it's effectively a knockout, or you have an overexpression, which both can be deleterious to development. [Image showing how pluripotent stem cells from different sources can be used to treat the human body highliting uniperental cells. The diagram specifically is highlighting ethics, safety, and preventing rejection] So we've been looking at uniparental stem cells, because they for the reason that even though that embryos don't develop, they actually can form a lot of tissues. So if you take the embryonic stem cells from these parthenogenetic or androgenetic embryos, because they develop to up to nine days or five days, so you can make embryonic stem cells from them, no problems. So you have these embryonic stem cells that can't form whole embryos. Is there a problem? No, don't hear me? No, okay, so the embryonic stem cells, you can inject them into blastocysts, like when people make knockouts, and you get these chimeras. So it's a combination of two cell types, and the wild-type cells support the androgenetic or the parthenogenetic cells to develop. So, you can actually get it, and if you do analysis on these chimeras, you get quite a lot of tissues that develop. Although the androgenetic cells alone tend to proliferate rapidly, which is not surprising because they have two copies of IGF2, which is a potent mitogen, and this actually contributes to malignancy, and the parthenogenetic cells have a slower development. And this is also reflected in the embryonic cancers that arise from consensus that are either androgenetic or parthenogenetic. So you can get ovarian teratomas from spontaneous parthenogenesis. So, spontaneous parthenogenesis in humans, which is fairly benign, and androgenetic fetuses or two paternal genomes, one maternal genome, form a choriocarcinoma, which is extremely aggressive. [Text on screen Paternal and maternal genomes confer opposite effects on proliferation, cell-cycle length, senescence, and tumor formation. The articles many authors are also listed. Primary Embryonic Fibroblast cultures from uniparental chimeras AG -> rapid proliferation -> malignancy / choriocarcinoma PG -> retarded proliferation -> early senescence / ovarian teratoma] So there's obviously strong limitations with these cells. And this is a quote here, which sums up the kind of premise for the work we've been doing. And that is that they don't think they're likely to be in use because of the imprinting or the genomic imprinting of these genes that are expressed abnormally, overexpressed, or not expressed at all. [Text on screen Can we use uniparental ES cells therapeutically? "..the recent production of parthenogenetic human ES cell lines is of note, although clinical application would likely be compromised by abnormal (maternal) genomic imprinting across all loci" Norio Nakatsuji, Fumiaki Nakajima & Katsushi Tokunaga. Nature Biotechnology 2008] So to test this hypothesis, we took a model where we generated chimeras from both parthenogenetic and antigenetic ES cells and took the fetal liver, which is an organ of hematopoiesis during development. And take the fetal liver, and we inject it into a lethally irradiated mice to see if the fetal liver or the embryonic blood system could replace the hematopoietic system of adult mice. So it's a functional assay. [Text on screen: Model illustrating hematopoietic reconstitution with ES cell-derived fetal cells] [Image on screen Es cells -> uniperental ES cels -> fetal chimera circa 13.5 dpc -> transplantation of fetal liver cells -> lethally irradiated recipitent featuring a mouse being injected.] And what we found is that these curves, you don't need to worry about interpreting them. But for both the androgenetic and the gynogenetic or the parthenogenetic cells, and because of the strains we use, we actually ended up with mice that had blood that was totally dry from the uniparental cells. So 100% androgenetic, 100% parthenogenetic in the blood. So even though they can't form whole embryos they can form whole organ systems, which is therapeutically applicable. [Graphs on screen Graph 1: Shows the percentage of blood cell reconstitution over time (months post-transplant) using Normal embryonic stem (ES) lines, labeled as “N.” Graph 2: Displays the same relationship using AG ES line 1, a uniparental embryonic stem cell line derived from one genetic parent. Graph 3: Shows data for GG ES line 1, another type of uniparental ES line, across multiple recipients post-transplant.] And this is just a lifespan, so there was no defects that happened long-term. So the mice will develop quite normally with its blood in their system. [Graph on-screen illustrates the health and longevity of recipients with uniparental hematopoiesis, studying both pre-transplantation and post-transplantation] So we also proved this therapeutically, because you can't make human chimeras. So we can do this totally in vitro in the dish. So we can take the ES cells, throw certain growth factors on there, and stimulate developmental pathways, and get blood cells from the tissue culture dish and reconstitute adult animals just as well. So that works very nicely. [Image showing the process of stem cells are modified into blood-forming cells and transplanting into a mouse] So, we wanted to look at why these differences occur. And obviously, there's gene expression differences, but there's also epigenetic marks. In this case, we're looking at methylation, which is differential between. This is a wild type, so 50% of the cell, the strands of DNA are marked, because they're from the maternal genome, and 50% are unmarked because they're from the, sorry, that's the paternal genome; 50% are unmarked, because they're from the maternal genome. And if we look at the cells we get from the blood from these animals, they still maintain their imprinting. So, the imprinting doesn't seem to have any role in the blood development. [Text on screen: Parent-of-origin methylation at the lgf2/H19 ICR is retained in adult engrafted uniparental cells.] [Graph showing that a specific DNA methylation pattern from one parent is preserved in adult stem cells after they are transplanted] So we have two hypotheses: either imprinting is not relevant or required in the blood, or there's selection of adaptive clones. So there's clonal selection of the cells we're putting in, which are precursors or stem cells that select out the ones that are viable or mutated somehow, and these manage to populate abnormally, what's going on? [Text on screen Result Interpretations... 1. Genomic imprinting is not relevant or required in HSC or mature blood - & or 2. Selection of adaptive clones during differentiation/engraftment overcomes restrictions imposed by genomic imprinting] So what we're looking at at the moment, one of the projects we're doing is we have done taking the blood cells from these two different animals, and we've done differential genetic expression analysis. And we have identified novel and printed genes which are potentially interesting in development of various systems. So one of the rotation projects that people do in my lab is they come and they take one or two of these genes and they analyze them for expression in various tissues and try to work out what their role is in development. And also, what's more relevant is what are they doing in adults. So if you have an imprinted gene which is defectively imprinted in an adult tissue, it's very well known that it has a lot of involvement in tumor genesis. So we have a whole list of genes which we've looked at, and one of the organs that's most interesting for imprinting abnormalities is the brain. And here's a list of genes and syndromes that are truly well-established and proven to be involved in disease. [Text on screen Imprinting involvement in neural development and behavior -Angelman syndrome: mental retardation, ataxia, seizures, "happy disposition", lack of maternally expressed genes including UBE3A -Prader Willi Syndrome: hypotonia, compulsive eating, behavioral abnormalities, mild mental retardation, lack of paternally expressed genes including Necdin -Ub23A(red): mice lacking maternal allele exhibit learning deficits GnasxL and Peg3 (blue): paternally expressed genes; affect suckling behavior or pups Magel2(blue): circadian rhythm Murine PG-N chimeras: aggressive behavior positively correlates with PG cell contribution PG/GG ES cells(red): some aspects of neural differentiation capacity studied AG ES cells(blue): unknown] So we have done some tissue transplant models in rats. So we've generated lesions, and we've transplanted androgenetic cells into the brains of rats, see if they integrate. That's what project I'm doing in collaboration with a group in Germany. [Text on screen Stereotactic transplantation of ESC-derived pan-neural progenitor cells (pNPCs) into the stratum of a mouse traumatic brain injury model Procedure of transplantation experiment: 1. A cryolesion was placed on the right parietal cortex. 2. After 10 days post-lesion, pNPCs were transplanted into striatum. (coordinates: AP +1.0 mm, ML +1.5 mm from bregma, DV – 2.5 mm from dura) (donor cells are in vitro differentiated from eGFP-positive ESCs.) 3. At 4 weeks post-transplant, murine brains were isolated and sagittal sectioned. To analyze donor cells, brain sections were stained using anti-eGFP and neural-specific antibodies] [Image on screen: Mouse brain with labeled region of injection and region of lession on the right hand side of the brain.] And we also have another model currently ongoing, which is funded by AR1, and that is the FAH model, which is an enzyme deficiency. And this is a great model because it's a mouse that will die after birth, several days as a neonate, if it doesn't have this enzyme, okay, because there's a buildup of tyrosinase pathway metabolites unless you give them this drug, NTBC, which is very expensive. And if we inject them with this drug, we can induce, we can stop the buildup of reagents. So if we have a successful transplant, where you have these patches here represent wild-type liver cells in the liver, as it grows, we can tease these cells to grow more and more, take over the liver, and give it a normal metabolism. [Text on screen: In vivo selection of Fah-expressing hepatocytes in the Fah (-/-) mouse model.] So again, it's a die or live assay for the potency of the stem cells we transplant. We're doing that with androgenetic and parthenogenetic cells. So the other aspect of my lab is that maybe you don't wanna do anything with imprinting, but we run the transgenic mouse core, so we have a lot of tools and technology. So if you're interested in going to another lab, for example, that does working with mouse knockouts or wants to generate any kinda special model or chimera model, then rotation in our lab could be useful. Because you can learn these other techniques, which make you very attractive to other labs as a rotation student. [Text on screen Transgenic and Embryonic Stem Cell Services -ES Cell Targeting In Vitro -Chimeras for germline transmission of targeted ES cells -Conditional Knockout Mice (Cre or FLP Electroporation) -Embryonic Stem Cell Line Derivation -Pronuclear Injection -Rederivation -Inter-Vivarium Transfer -Embryo Cryopreservation -Sperm Cryopreservation -Training] So if you have any questions, you can find my name on the web, cause you're all competent in finding faculty emails.