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Mike Gallagher

@geneticsmike7.bsky.social
179 followers 225 following 430 posts

Geneticist and stem cell biologist studying epigenetics and neurodegeneration at Whitehead Institute and MIT.

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Mike Gallagher @geneticsmike7.bsky.social · 02/11/2025
19/ strangely, leaky SALL1 is not due to unwanted splicing in the absence of drug, but rather the unspliced pre-mRNA somehow escapes the nucleus and is translated in-frame to make the full-length protein. We still don't know how this works.
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Mike Gallagher @geneticsmike7.bsky.social · 02/11/2025
17/ we also show that we can get inducible gene editing with Xon hPSCs, but only if sgRNAs are introduced transiently (panels C-E). Cas9 must have enough leakiness for maximal editing with constitutive lentiviral sgRNA introduced into hPSCs (A-B)
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Mike Gallagher @geneticsmike7.bsky.social · 02/11/2025
16/ what about other genes? Here we show Xon works for inducible control of GRN expression, and we can overexpress GRN in iPSC-derived microglia, which already express boatloads of it. P2A peptides improve both GRN and SMAD, and importantly reduce N-terminal tag to a single aa.
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Mike Gallagher @geneticsmike7.bsky.social · 02/11/2025
14/ a note of caution is that while background EGFP is minor compared to even the lowest drug induced expression levels, this may need mitigation when using TFs or other strong cell state regulators
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Mike Gallagher @geneticsmike7.bsky.social · 02/11/2025
13/ Here's the commonly used H1 ESC background, differentiated into microglia with an embryoid body protocol (we used EB and monolayer)
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Mike Gallagher @geneticsmike7.bsky.social · 02/11/2025
12/ quantification shows how tunable this system really is! Below the 100nM dose, linear correlations between drug dose and EGFP is basically perfect both before and after differentiation
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Mike Gallagher @geneticsmike7.bsky.social · 02/11/2025
11/ again with AAVS1-targeted EGFP, what we saw was remarkable: first time we've ever seen robust inducibility after long differentiations, this time 4-7 weeks, across hPSC backgrounds and differentiation protocols!
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Mike Gallagher @geneticsmike7.bsky.social · 02/11/2025
9/ we also looked at leakiness, and leaky EGFP varied by system, with Tet-On being pretty tightly controlled (although silenced later on), and degrons showing variability. Note lack of degron tunability in panel C.
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Mike Gallagher @geneticsmike7.bsky.social · 02/11/2025
8/ We next tested degrons, as this have been used successfully for transgene expression specifically in fully differentiated cells. However, their leakiness, tunability, and inducibility hasn't been thoroughly tested.
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Mike Gallagher @geneticsmike7.bsky.social · 02/11/2025
7/ all approaches used EGFP after AAVS1 safe harbor targeting. While these systems worked in hPSCs (albeit with slower kinetics), they still got silenced during differentiation
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Mike Gallagher @geneticsmike7.bsky.social · 02/11/2025
6/ we then tried to fix this by adding a Tet-Off-TET1 fusion protein, as DNA methylation has been proposed to underlie Tet-On silencing. Here's the idea, with existing systems (top) getting silenced before DOX is added, and new systems (bottom) we hoped would fix it
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Mike Gallagher @geneticsmike7.bsky.social · 02/11/2025
5/ First we showed that Tet-On-based transgenes are silenced early during microglia or macrophage differentiation, regardless of transgene delivery method, transgene components, hPSC background, or differentiation protocol
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Mike Gallagher @geneticsmike7.bsky.social · 22/06/2025
29/ Finally, we compared these DEGs with those from MS patient microglia from gray matter, white matter, active/inactive lesions, etc. We found a significant overlap, suggesting that iPSC-microglia can recapitulate some MS-relevant pathways when perturbing risk genes at individual loci.
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Mike Gallagher @geneticsmike7.bsky.social · 22/06/2025
28/ we also find striking concordance between DEGs across these 5 loci - many genes were shared and directionally concordant. Homeostatic microglia pathways such as GTPase activity were downregulated in cells with MS risk gene perturbation, whereas oxphos and other metabolic pathways were increased.
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Mike Gallagher @geneticsmike7.bsky.social · 22/06/2025
27/ this wasn't just due to altering microglia genes in general, as AD heritability was not nearly as enriched, despite the strong connection of AD SNP heritability with microglia. The effect persisted when excluding the cis target gene and HLA.
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Mike Gallagher @geneticsmike7.bsky.social · 22/06/2025
25/ and three other loci: CD86, IRF5, and SLC25A19, the latter of which we show GRB2 as the likely SNP target gene
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Mike Gallagher @geneticsmike7.bsky.social · 22/06/2025
24/ complement C3, which is regulated by upstream enhancers with MS SNPs, but no effect on TNFSF14 which is actually closer to the GWAS signal......
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Mike Gallagher @geneticsmike7.bsky.social · 22/06/2025
23/ we identified the causal MS risk gene at 5/9 loci, including MERTK, a well know regulatory of phagocytosis...
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Mike Gallagher @geneticsmike7.bsky.social · 22/06/2025
22/ when we performed the screen, the microglia were very homogeneous, and even lacked markers of highly similar border-associated macrophages like LYVE1. We used MOI of 1.5, sequenced ~120K cells with ~10B read pairs, got ~400 cells/guide and ~2K genes/cell.
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Mike Gallagher @geneticsmike7.bsky.social · 22/06/2025
21/ Vpx lentiviral system to a Perturb-seq vector for efficient, non-toxic, minimally-activating CRISPRi in #microglia, using dCas9-KRAB with ZIM3 KRAB domain:
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Mike Gallagher @geneticsmike7.bsky.social · 22/06/2025
19/ a really interesting locus if WWOX/MAF, which has both AD and MS GWAS SNPs, as well as MS outside variants, overlapping microglial enhancers spanning 500kb. PLAC-seq in ex vivo microglia and PCHi-C in iPSC-microglia really suggest MAF as the target gene here
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Mike Gallagher @geneticsmike7.bsky.social · 22/06/2025
18/ so here's what we get when we apply this approach to all 200 non-MHC loci. Outside variant + GWAS SNP enrichments in enhancers suggest that some loci can be resolved to a single cell type of origin, including 17 plausible microglial effect loci.
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Mike Gallagher @geneticsmike7.bsky.social · 22/06/2025
16/ the way it works is we 1) look at each GWAS locus, 2) identity putative enhancers that interact with the same target gene as the GWAS region, and 3) determine if there are any SNPs in these "outside" regions that conspire with the GWAS SNPs to further alter risk.
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Mike Gallagher @geneticsmike7.bsky.social · 22/06/2025
13/ The BIN1 AD risk locus is well characterized, and our iPSC-microglia seem to do a good job recapitulating the epigenomic patterns here.
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Mike Gallagher @geneticsmike7.bsky.social · 22/06/2025
12/ while we focused on microglia for the rest of the study, here are the s-LDSC results for NPCs, neurons and astrocytes - surprisingly no astrocyte enriched traits!
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Mike Gallagher @geneticsmike7.bsky.social · 22/06/2025
11/ similar to global ChIP-seq, s-LDSC also showed strongest concordance between iPSC and ex vivo microglia, with top disease SNP enrichments shown on left. Enhancers only seen in ex vivo cells (right) did not show any enrichments, so our #microglia seem to be pretty physiological here.
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Mike Gallagher @geneticsmike7.bsky.social · 22/06/2025
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Mike Gallagher @geneticsmike7.bsky.social · 22/06/2025
9/ but we really want to know if the epigenome of iPSC-derived cells allow for mechanistic studies of GWAS SNPs, so we did H3K27ac ChIP-seq and compared the data to those of "ex vivo" uncultured primary neurons and glia, and performed s-LDSC analyses on 55 diseases/traits
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Mike Gallagher @geneticsmike7.bsky.social · 22/06/2025
8/ neurons were synaptically active, microglia phagocytosed and too up lipids....
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Mike Gallagher @geneticsmike7.bsky.social · 22/06/2025
7/ etc. etc.
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Mike Gallagher @geneticsmike7.bsky.social · 22/06/2025
6/ RNA-seq looked pretty good, we were getting the right cell identities....
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Mike Gallagher @geneticsmike7.bsky.social · 22/06/2025
5/ so can we use iPSC-derived #neurons #astrocytes #microglia to better understand these loci, and how do these models need to be improved? We made these cell types from female and male iPSCs and did a bunch of QC
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Mike Gallagher @geneticsmike7.bsky.social · 22/06/2025
4/ and more recently from a review by Alsheikh et al, BMC Med Genomics....not much improvement!
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Mike Gallagher @geneticsmike7.bsky.social · 22/06/2025
3/ epigenetic data, appropriate experimental models, and high-throughput perturbations. Here's what post-GWAS mechanistic studies looked like during my Ph.D....
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Mike Gallagher @geneticsmike7.bsky.social · 01/12/2024
Looks like R01s go as far as they used to?
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