Posters

Fragile X syndrome (FXS), the most prevalent inherited intellectual disability, is caused by loss of fragile X translational regulator 1 (FMR1).We have previously shown that FMR1 represses the levels and activities of ubiquitin ligase MDM2 in young adult FMR1 mutant mice and treatment by MDM2 inhibitor Nutlin-3 rescues both hippocampal neurogenic and cognitive deficits in FMR1-deficient mice when analyzed within one month after treatment, a time period need for adult new neuron development and integration. However, it is unknown whether Nutlin-3 treatment can have long-lasting therapeutic effects. Here, we show that treatment with Nutlin-3 of 2-month old young adult FMR1-deficient mice prevents the emergence of neurogenic and cognitive deficits in mature adult FXS mice at 6 months of age. To explore the molecular mechanisms underlying the long-lasting therapeutic effect of Nutlin-3, we performed comprehensive transcriptome analysis of hippocampus of FMR1-deficient and control mice treated with either Nutlin-3 or vehicle. Our findings suggest that impaired neurogenesis and cognitive functions in FXS at mature adult ages may be prevented by early intervention by Nutlin-3 treatment.

Epicardial cells (EpiCs) are necessary for proper heart formation, however little is known about crosstalk between EpiCs and cardiomyocytes (CMs) during development and the potential impact of EpiCs on CM maturation. To investigate the effects of EpiCs on CM commitment and maturation, we differentiated human pluripotent stem cells (hPSCs) to cardiac progenitor cells (CPCs) and EpiCs, and cocultured EpiCs and CPCs for two weeks. We identified changes in CM structural maturation including sarcomere organization and induced proliferation, and we demonstrated that these changes are only observed in direct coculture and that conditioned medium or indirect coculture is insufficient. Finally, single cell sequencing of EpiC cocultures had bi-directional effects and biased EpiC lineages. This work suggests important crosstalk between EpiCs and CMs during differentiation can be used to influence cell fate and improve the ability to generate cardiac cells and tissues for in vitro models and development of cardiac cellular therapies.

In vitro human pluripotent stem cell (hPSC)-derived blood-brain barrier (BBB) models hold great potential for studying developmental processes due to cellular components with embryonic-like differentiation states. We use multicellular hPSC-derived BBB models to investigate cellular signaling inputs and pathways vital to barrier property specification in brain endothelium during development and to more accurately model the BBB in vitro. We specifically explore the inductive effects of hypoxia, a crucial environmental factor in the developing CNS, on these signaling networks and endothelial barrier property acquisition. Hypoxia was either mimicked with cobalt chloride (CoCl2), a HIF-1α stabilizer, or induced with 5% O2. Immunostaining and flow cytometry of hPSC-derived endothelial progenitors (EPCs) differentiated for 5 days to naïve endothelial cells (ECs) with CoCl2 show increased (P<0.005) expression of BBB-specific glucose transporter GLUT-1. Factors secreted by neural progenitor-like neural rosettes exposed to 5% O2 also contribute to halved (P<0.01) hPSC-EC expression of caveolin-1, a vesicle-forming protein responsible for transcytosis (a feature of non-selective endothelium).

The blood-brain barrier (BBB) is critical for neurological health, and BBB breakdown has been linked to multiple diseases. The BBB consists of brain microvascular endothelial cells (BMECs), whose barrier properties are induced and maintained by adjacent cell types such as brain pericytes. Previous studies showed that the brain pericytes are required for both formation and maintenance of the BBB. Furthermore, it was found that pericytes are able to degrade proteins that can accelerate neurovascular damage, thus BBB breakdown may deteriorate in case of pericyte malfunction or deficiency. To investigate effects of brain pericytes in BBB maintenance or breakdown, we started by culturing human pluripotent stem cell (hPSC)-derived BMEC-like cells and brain pericyte-like cells together with human-sourced serum proteins in the media. We found that the tightness of the barrier formed by BMECs decreased dramatically after the pericytes were removed from the culture but could be partially rescued by pericyte-conditioned medium. Our work suggests that the BBB deterioration in the presence of serum proteins and the absence of brain pericytes can be recapitulated by hPSC-derived models.

Engineered tissues are a promising treatment for functional cardiac restoration to replace dead cardiomyocytes (CMs) after heart attack. Using protocols developed by Dr. Palecek’s lab, human pluripotent stem cells (hPSCs) provide a limitless supply of cardiac cells. To mimic the native heart environment, and create more mature cardiac tissues, we have co cultured CMs with other cardiac cell types. Epicardial cells (EpiCs) are of particular interest since they aid in cardiac development and repair. To assess interactions between EpiCs and developing CMs, we cocultured EpiC with developing CMs for two weeks in the presence of a TGFβ inhibitor to prevent further differentiation of EpiCs. We observed significantly higher CM Ki67 expression in EpiC cocultures, indicating CM proliferation, and significantly higher CM MLC2v+ expression in EpiC cocultures, indicating CM ventricular specification. Even so, we found no change in markers of CM maturation FSC-A or cTnI, in EpiC cocultures. Next, we confirmed CM proliferation is dose dependent on its exposure to EpiC. This study identifies noteworthy EpiC-CM interactions to be used to further develop mature hPSC-CMs for eventual widespread use.

Sarcopenia, the degeneration of muscle mass and function with age, currently lacks effective prevention and treatment. To better understand muscle aging, in vitro experiments utilizing human pluripotent stem cell(hPSC)-derived myogenic progenitors were performed to study non-cell-autonomous aspects of skeletal muscle cell aging. Previously, treatment with pooled sera from old and young rats showed that old sera decreased whereas young sera increased expansion rate and muscle differentiation of hPSC-derived myogenic progenitors in culture. This experiment sought to verify the results using human plasma pooled from 6 young and 6 old female donors. Consistent with previous results, compared to hPSC-derived myogenic progenitors treated with young plasma, treatment with old plasma caused a reduced growth rate at undifferentiated stage and impaired myotube formation following terminal differentiation. These results imply that the systemic biokinetics of aged individuals contains components that impair self-renewal capacity and differentiation potential of myogenic progenitors. More insights into this mechanism would help improve the therapeutic approaches for age-related muscle wasting.

Our service provides expertise to generate induced pluripotent stem cells (iPSC) by reprogramming and to perform iPSC gene editing by CRISPR/Cas9. We serve the UW-Madison human stem cell community, allowing scientists to focus their time and resources on the biological study and therapeutic applications of PSCs. To learn more, visit https://www.waisman.wisc.edu/ipsc-services. To request services, please send email to pjiang39@wisc.edu.

Alzheimer’s Disease (AD) is a neurodegenerative disease that leads to dementia, loss of memory, and other behavioral abnormalities. Recently, evidence from post-mortem tissue analysis and mouse studies have suggested that blood-brain barrier (BBB) dysfunctions may contribute to AD pathogenesis. From a proteomics analysis comparing AD and control patients’ post-mortem brain microvessles, we discovered several target genes that are significantly upregulated or downregulated in AD samples. Thus, to study the mechanism leading to BBB changes in AD, we established PiggyBac-based inducible CRISPRi and CRISPRa systems in induced pluripotent stem cells (iPSCs) and differentiated the iPSCs to brain microvascular endothelial cells (BMECs). Lentiviruses were used to deliver sgRNAs targeting different AD-related genes in BMECs. Preliminarily, we validated the functionality of CRISPRi and CRISPRa systems both in iPSCs and differentiated BMECs. Future efforts will focus on recreating AD-related phenotypes in BMECs and exploring the molecular mechanism behind these changes.

Hematopoietic stem and progenitor cells (HSPCs) can differentiate into all blood cells. Improving the differentiation in vitro will enable unlimited supplies of HSPCs for cell therapies targeting bone marrow-related diseases. This study identifies inefficiencies in the ESC to HSPC differentiation in vitro. On day 0, cells are plated on low-adherent plates and incubated at 5% oxygen1. From day 2 to 8, cytokines are added to help cells towards hematopoietic potential. On day 8, magnetic-activated cell sorting (MACS) obtained CD34+ cells. The yield was 8.5%, 7%, and 5.5% in 3 replicates. Cells were plated on a Matrigel coated plate for the endothelial to hematopoietic transition (EHT) and flow cytometry was conducted. There was 8.76% CD34 expression pre-MACs and 100% CD34 expression post-MACS, consistent with day 8 cell counts. However, cells did not adhere to the plate. The potential problem is the Matrigel concentration which may be altering the cell’s potential to adhere to the plate and go through EHT. Future studies should explore Matrigel concentrations and the optimization of cell viability for cryopreservation of HSPCs.

Given the limited ability to study early human embryonic development, there exists a demand for model systems that recapitulate early human developmental events in vitro. It is found that when human pluripotent stem cells are cultured in neural progenitor media, they spontaneously form polarized rosette structures similar to the developing neural tube. Current techniques result in nascent tissue morphologies incapable of modeling beyond the progenitor stage. This study aimed to optimize conditions for inducing tissue maturation via controlled outgrowth on micropatterned culture substrates. Microcontact printed substrates, which spatially isolate singular neural rosettes, were conjugated with an adhesion-promoting peptide, RGD or heparin binding peptide, via click chemistry. Rosettes introduced to RGD experienced the most extensive outgrowth. In-situ functionalization of microprinted substrates provides a feasible means of micropatterning hPSC-derived neural tissue morphology with spatiotemporal control. The ability to augment this platform with such chemistries provides an interface to study early neural development as well as identify alternative growth-promoting peptides.

To regeneratively heal heart disease, scalable iPSC-CM manufacturing must adhere to FDA regulations, which cite critical quality attributes (CQAs) to be used for real time monitoring. Cell confluency is widely cited as a CQA in stem cell protocols but is unexamined in context of iPSC-CM differentiation efficiency. By using PHANTAST cell segmentation software, parameter sets were tuned to accurately quantify this metric. Using Brightfield microscopy datasets of cell culture plates, reference sheets were made to aide estimation of confluency in lab. Further, 3 passages of stem cells were imaged every 4 hours to Day 15 during CM differentiation. Using segmentation, the confluency at notable “inflection points” of cell migration schemes were compared to typical CQAs using linear and nonlinear approaches. Variable presence charts of nonlinear algorithmic data modeling revealed confluency’s power in prediction of CM differentiation efficiency.

The combinatorial output of epigenetic modifications with transcription factors controls gene expression. In most cell types, gene expression is fine tuned for a specific functional output. By contrast, pluripotent stem cells (PSCs) are poised to express genes for any specialized cell type despite their self-renewal. These properties are enabled by an open chromatin structure enriched for transcriptional activation associated histone modifications such as acetylation. Surprisingly, PSCs are depleted for histone modifications associated with transcriptional elongation, most notably H3K79me2. The 8-fold difference in H3K79me2 occurs at shared locations in both somatic and pluripotent cells at highly transcribed genes. Inhibition of the H3K79methyltransferase, Dot1L, enhances acquisition of pluripotency without suppressing the somatic transcriptome. Instead, remarkably, disruption of Dot1L promotes acetylation of H3 and H4. H3K9ac is specifically increased at genes with a rapid transcriptional elongation rate. Thus reduction of H3K79 methylation enables the generation of a hyperacetylated epigenome that is the cornerstone of pluripotency.

Glial fibrillary acidic protein (GFAP) is an intermediate filament that is enriched in astrocytes. Altered GFAP levels have been shown in multiple CNS disorders, e.g. Alzheimers’ disease, and correlate with cognitive decline. In addition, point mutations of GFAP cause Alexander Disease (AxD), which is a rare yet fatal neurodegenerative disease. Therefore, GFAP likely plays an important role in the CNS, yet, its functions are largely unknown. Previous studies from our lab using hiPSC-derived astrocytes imply the involvement of GFAP in vesicular trafficking. Continuing the study, we first show that GFAP forms direct contacts with ER and Golgi. Using TurboID mediated proximity ligation assay, we systematically examined proteins that interact with GFAP, and found a large battery of proteins involved in vesicular trafficking. Further, we find that GFAP knock-out (KO) alters the morphologies of ER and Golgi and alters protein trafficking. These results suggest that GFAP play an important role in secretory pathway. Since secreted factors from astrocytes are critical for neuronal survival and functions, this may explain the non-cell autonomous neuronal degeneration in AxD.

Hox genes encode transcription factors that are critical for embryonic skeletal patterning and organogenesis. The Hoxa5, Hoxb5 and Hoxc5 paralogs are expressed in the lung mesenchyme and function redundantly during embryonic lung development. Conditional loss of function of these genes during early postnatal stages leads to severe defects in alveologenesis, specifically in the generation of the elastin network. Here we show the surprising results that that mesenchyme-specific loss of Hox5 function at adult stages leads to rapid disruption of the mature elastin matrix, alveolar enlargement, and an emphysema-like phenotype. As the elastin matrix of the lung is considered highly stable, an adult disruption of the matrix was not predicted. Just two weeks after deletion, adult Hox5 mutant animals show significant increase in alveolar space and changes in pulmonary function, including reduced elastance and increased compliance. Examination of the ECM of adult Tbx4rtTA;TetOCre;Hox5afafbbcc lungs demonstrate a disruption of the elastin network. In culture, fibroblasts from Hox5 mutant lungs exhibit reduced adhesion.

Limited regeneration capacity of mammals causes the permanent fibrotic tissue in the damaged heart, leading to cardiac dysfunction. In contrast, regenerative species, like zebrafish, exhibit the robust heart regeneration along with scar resolution. However, signaling pathways influencing cardiac regeneration program and fibrosis remain elusive. Here, we show that Interleukin-11 (Il-11) signaling regulates both cardiac regeneration and fibrosis. Expression analysis of the il11a knock-in reporter line revealed that il11a is robustly induced upon cardiac injury and peaked at the early regenerative phase. The loss-of-function study indicated that il11a is a key regenerative factor for the hearts. Our gain-of-function study and transcriptomic analysis demonstrated that conditional il11a overexpression in the myocardium of uninjured hearts can activate cardiac regeneration programs. Interestingly, we also found that the prolonged expression of il11a in both uninjured and injured hearts causes collagen-rich scarring, indicating detrimental effects of il11a on regeneration. Overall, our findings unveil the conflicting role of Il-11 signaling in heart regeneration.