| 1. |
- De Genst, Erwin, et al.
(författare)
-
Blocking phospholamban with VHH intrabodies enhances contractility and relaxation in heart failure
- 2022
-
Ingår i: Nature Communications. - Stockholm : Karolinska Institutet, Dept of Cell and Molecular Biology. - 2041-1723.
-
Tidskriftsartikel (refereegranskat)abstract
- The dysregulated physical interaction between two intracellular membrane proteins, the sarco/endoplasmic reticulum Ca2+ ATPase and its reversible inhibitor phospholamban, induces heart failure by inhibiting calcium cycling. While phospholamban is a bona-fide therapeutic target, approaches to selectively inhibit this protein remain elusive. Here, we report the in vivo application of intracellular acting antibodies (intrabodies), derived from the variable domain of camelid heavy-chain antibodies, to modulate the function of phospholamban. Using a synthetic VHH phage-display library, we identify intrabodies with high affinity and specificity for different conformational states of phospholamban. Rapid phenotypic screening, via modified mRNA transfection of primary cells and tissue, efficiently identifies the intrabody with most desirable features. Adeno-associated virus mediated delivery of this intrabody results in improvement of cardiac performance in a murine heart failure model. Our strategy for generating intrabodies to investigate cardiac disease combined with modified mRNA and adeno-associated virus screening could reveal unique future therapeutic opportunities.
|
|
| 2. |
- Foo, Kylie S, et al.
(författare)
-
Human ISL1+ ventricular progenitors self-assemble into an in vivo functional heart patch and preserve cardiac function post infarction
- 2018
-
Ingår i: Molecular Therapy. - Stockholm : Karolinska Institutet, Dept of Cell and Molecular Biology. - 1525-0016. ; 26:7, s. 1644-1659
-
Tidskriftsartikel (refereegranskat)abstract
- The generation of human pluripotent stem cell (hPSC)-derived ventricular progenitors and their assembly into a 3-dimensional in vivo functional ventricular heart patch has remained an elusive goal. Herein, we report the generation of an enriched pool of hPSC-derived ventricular progenitors (HVPs), which can expand, differentiate, self-assemble, and mature into a functional ventricular patch in vivo without the aid of any gel or matrix. We documented a specific temporal window, in which the HVPs will engraft in vivo. On day 6 of differentiation, HVPs were enriched by depleting cells positive for pluripotency marker TRA-1-60 with magnetic-activated cell sorting (MACS), and 3 million sorted cells were sub-capsularly transplanted onto kidneys of NSG mice where, after 2 months, they formed a 7 mm x 3 mm x 4 mm myocardial patch resembling the ventricular wall. The graft acquired several features of maturation: expression of ventricular marker (MLC2v), desmosomes, appearance of T-tubule-like structures, and electrophysiological action potential signature consistent with maturation, all this in a non-cardiac environment. We further demonstrated that HVPs transplanted into un-injured hearts of NSG mice remain viable for up to 8 months. Moreover, transplantation of 2 million HVPs largely preserved myocardial contractile function following myocardial infarction. Taken together, our study reaffirms the promising idea of using progenitor cells for regenerative therapy. Correction in Mol Ther. 2021 Jan 6;29(1):409, DOI: 10.1016/j.ymthe.2020.11.015
|
|
| 3. |
- Foo, Kylie S
(författare)
-
Distribution and connectivity of messenger molecules in the control of energy metabolism : focus on neuropeptides and calcium binding proteins
- 2010
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Feeding is an essential and complex behavior which aims to provide the energy required for maintaining physiological homeostasis. The drive to feed is a powerful stimulus arising from metabolic demands, and reinforced by evolutionary pressure. The current epidemic in obesity, and associated disorders such as diabetes, makes it clinically vital to understand the mechanisms behind the control of energy metabolism. Feeding is a process governed by the central nervous system (CNS); particularly through the interplay between different hypothalamic nuclei. At the heart of the feeding neuro-circuitry lies the arcuate nucleus (ARC) which acts as a metabolic sensor, taking stock of the supply and demands of energy in the body, and coordinating food intake and energy expenditure. The work in this thesis aimed to explore the neuro-anatomical substrate of metabolic control, and the mediators involved. The ARC contains two distinct sets of functionally antagonistic neurons. One group of neurons express the orexigenic peptides, neuropeptide Y (NPY) and agouti gene related peptide (AGRP); while the other set expresses the anorexigenic peptides, proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript. In paper V, we describe the histochemistry of NPY/AGRP and POMC neurons with regard to their anatomical interrelationship at the cell body and terminal level. A common experimental problem is that the ARC NPY cell bodies are difficult to distinguish and visualize in electrophysiological experiments and for immunohistochemistry. Therefore, in paper III, a novel transgenic mouse which expresses bright Renilla green fluorescent protein in NPY neurons was generated. Using this model, a comprehensive map of NPY-expressing cells in the CNS was generated and the effects of the satiety-inducing gut-brain bombesin peptides on ARC neurons were explored. Bombesin was found to exert powerful depolarizing actions on NPY and POMC neurons alike. Calcium binding proteins (CaBPs) have been used extensively to delineate neuronal populations, but the ARC has not yet been subjected to such analysis. In Paper IV we show that three major CaBPs (calbindin D-28k, calretinin, and parvalbumin) are all expressed in the ARC, but displayed little co-localization with previously described cell groups. One exception was POMC neurons, of which distinct subpopulations stained for calbindin D-28k and calretinin, respectively. Another CaBP, nucleobindin 2 (NUCB2; also known as nesfatin), has recently been proposed as a central anorexigenic mediator. In Paper I, the CNS distribution of this protein was shown to include nuclei that participate in all three output channels of metabolic control, i.e. behavioral, endocrine and autonomic modulation. Our data also suggest that NUCB2 may not act as a cleaved and secreted messenger as proposed, but rather may play an intracellular role. The wide distribution of NUCB2 in the neuroendocrine system prompted us to explore this protein in the pancreas (Paper II). We show that NUCB2 is exclusively expressed in insulin-producing β cells, and that islet NUCB2 is dramatically decreased in the diabetic Goto-Kakizaki rat, an effect that is normalized by fasting. These data indicate that NUCB2 may play a role in metabolic control also outside of the CNS.
|
|
| 4. |
- Poch, Christine M, et al.
(författare)
-
Migratory and anti-fibrotic programmes define the regenerative potential of human cardiac progenitors
- 2022
-
Ingår i: Nature Cell Biology. - Stockholm : Karolinska Institutet, Dept of Cell and Molecular Biology. - 1465-7392 .- 1476-4679.
-
Tidskriftsartikel (refereegranskat)abstract
- Heart regeneration is an unmet clinical need, hampered by limited renewal of adult cardiomyocytes and fibrotic scarring. Pluripotent stem cell-based strategies are emerging, but unravelling cellular dynamics of host–graft crosstalk remains elusive. Here, by combining lineage tracing and single-cell transcriptomics in injured non-human primate heart biomimics, we uncover the coordinated action modes of human progenitor-mediated muscle repair. Chemoattraction via CXCL12/CXCR4 directs cellular migration to injury sites. Activated fibroblast repulsion targets fibrosis by SLIT2/ROBO1 guidance in organizing cytoskeletal dynamics. Ultimately, differentiation and electromechanical integration lead to functional restoration of damaged heart muscle. In vivo transplantation into acutely and chronically injured porcine hearts illustrated CXCR4-dependent homing, de novo formation of heart muscle, scar-volume reduction and prevention of heart failure progression. Concurrent endothelial differentiation contributed to graft neovascularization. Our study demonstrates that inherent developmental programmes within cardiac progenitors are sequentially activated in disease, enabling the cells to sense and counteract acute and chronic injury.
|
|
| 5. |
- Xu, Jiejia, et al.
(författare)
-
Genome‐wide CRISPR screen identifies ZIC2 as an essential gene that controls the cell fate of early mesodermal precursors to human heart progenitors
- 2020
-
Ingår i: Stem Cells. - Stockholm : Karolinska Institutet, Dept of Cell and Molecular Biology. - 1066-5099 .- 1549-4918.
-
Tidskriftsartikel (refereegranskat)abstract
- Cardiac progenitor formation is one of the earliest committed steps of human cardiogenesis and requires the cooperation of multiple gene sets governed by developmental signaling cascades. To determine the key regulators for cardiac progenitor formation, we have developed a two-stage genome-wide CRISPR-knockout screen. We mimicked the progenitor formation process by differentiating human pluripotent stem cells (hPSCs) into cardiomyocytes, monitored by two distinct stage markers of early cardiac mesodermal formation and commitment to a multipotent heart progenitor cell fate: MESP1 and ISL1, respectively. From the screen output, we compiled a list of 15 candidate genes. After validating seven of them, we identified ZIC2 as an essential gene for cardiac progenitor formation. ZIC2 is known as a master regulator of neurogenesis. hPSCs with ZIC2 mutated still express pluripotency markers. However, their ability to differentiate into cardiomyocytes was greatly attenuated. RNA-Seq profiling of the ZIC2-mutant cells revealed that the mutants switched their cell fate alternatively to the noncardiac cell lineage. Further, single cell RNA-seq analysis showed the ZIC2 mutants affected the apelin receptor-related signaling pathway during mesoderm formation. Our results provide a new link between ZIC2 and human cardiogenesis and document the potential power of a genome-wide unbiased CRISPR-knockout screen to identify the key steps in human mesoderm precursor cell- and heart progenitor cell-fate determination during in vitro hPSC cardiogenesis.
|
|
