a-human-cerebral-organoid-model-of-neural-cell-transplantation

A Human Cerebral Organoid Model of Neural Cell Transplantation

The advancement of cell transplantation approaches requires model systems that allow an accurate assessment of transplanted cell functional potency. For ...

Université de Montréal

The small number of hematopoietic stem and progenitor cells in cord blood units limits their widespread use in human transplant protocols. We identified a family of chemically related small molecules that stimulates the expansion ex vivo of human cord blood cells capable of reconstituting human hematopoiesis for at least 6 months in immunocompromised mice. The potent activity of these newly identified compounds, UM171 being the prototype, is independent of suppression of the aryl hydrocarbon receptor, which targets cells with more-limited regenerative potential. The properties of UM171 make it a potential candidate for hematopoietic stem cell transplantation and gene therapy.

Cord blood expansion. Pyrimidoindole derivatives are agonists of human hematopoietic stem cell self-renewal.

Enhanced gene transfer efficiencies and higher yields of transplantable transduced human hematopoietic stem cells are continuing goals for improving clinical protocols that use stemcell-based gene therapies. Here, we examined the effect of the HSC agonist UM171 on these endpoints in both  and  systems. Using a 22-hr transduction protocol, we found that UM171 significantly enhances both the lentivirus-mediated transduction and yield of CD34 and CD34CD45RA hematopoietic cells from human cord blood to give a 6-fold overall higher recovery of transduced hematopoietic stem cells, including cells with long-term lympho-myeloid repopulating activity in immunodeficient mice. The ability of UM171 to enhance gene transfer to primitive cord blood hematopoietic cells extended to multiple lentiviral pseudotypes, gamma retroviruses, and non-integrating lentiviruses and to adult bone marrow cells. UM171, thus, provides an interesting reagent for improving the  production of gene-modified cells and for reducing requirements of virus for a broad range of applications.

UM171 Enhances Lentiviral Gene Transfer and Recovery of Primitive Human Hematopoietic Cells.

COVID-19 has severely impacted laboratory research. Analysis of the International Society for Stem Cell Research (ISSCR) member survey has highlighted a particular impact on clinical trials and early-career investigators. The stem cell community needs to support young researchers and ensure that stem cell medicine does not lose its momentum.

Survey Says: "COVID-19 Lockdown Hits Young Faculty and Clinical Trials".

Transfer RNA (tRNA) and their associated production and processing machinery can be coopted as a versatile tool for the production of guide RNAs (gRNAs) for Cas9-based genome engineering. Using different tRNA variants enables the production of gRNAs at a variety of steady state levels. Furthermore, engineered tRNAs can be used to process gRNAs from Pol-II transcripts, thus enabling spatial/temporal control of gRNA expression. Here we describe the design, cloning, and testing of tRNA scaffolds for both Pol-III-driven expression of different levels of gRNAs, and for processing gRNAs from Pol-II transcripts.

Harnessing tRNA for Processing Ability and Promoter Activity.

COVID-19 patients show heterogeneity in clinical presentation and outcomes that makes pandemic control and strategy difficult; optimizing management requires a systems biology approach of understanding the disease. Here we sought to potentially understand and infer complex disease progression, immune regulation, and symptoms in patients infected with coronaviruses (35 SARS-CoV and 3 SARS-CoV-2 patients and 57 samples) at two different disease progression stages. Further, we compared coronavirus data with healthy individuals (n = 16) and patients with other infections (n = 144; all publicly available data). We applied inferential statistics (the COVID-engine platform) to RNA profiles (from limited number of samples) derived from peripheral blood mononuclear cells (PBMCs). Compared to healthy individuals, a subset of integrated blood-based gene profiles (signatures) distinguished acute-like (mimicking coronavirus-infected patients with prolonged hospitalization) from recovering-like patients. These signatures also hierarchically represented multiple (at the system level) parameters associated with PBMC including dysregulated cytokines, genes, pathways, networks of pathways/concepts, immune status, and cell types. Proof-of-principle observations included PBMC-based increases in cytokine storm-associated IL6, enhanced innate immunity (macrophages and neutrophils), and lower adaptive T and B cell immunity in patients with acute-like disease compared to those with recovery-like disease. Patients in the recovery-like stage showed significantly enhanced TNF, IFN-γ, anti-viral, HLA-DQA1, and HLA-F gene expression and cytolytic activity, and reduced pro-viral gene expression compared to those in the acute-like stage in PBMC. Besides, our analysis revealed overlapping genes associated with potential comorbidities (associated diabetes) and disease-like conditions (associated with thromboembolism, pneumonia, lung disease, and septicemia). Overall, our COVID-engine inferential statistics platform and study involving PBMC-based RNA profiling may help understand complex and variable system-wide responses displayed by coronavirus-infected patients with further validation.

A blood transcriptome-based analysis of disease progression, immune regulation, and symptoms in coronavirus-infected patients.

A problematic feature of many human cancers is a lack of understanding of mechanisms controlling organ-specific patterns of metastasis, despite recent progress in identifying many mutations and transcriptional programs shown to confer this potential. To address this gap, we developed a methodology that enables different aspects of the metastatic process to be comprehensively characterized at a clonal resolution. Our approach exploits the application of a computational pipeline to analyze and visualize clonal data obtained from transplant experiments in which a cellular DNA barcoding strategy is used to distinguish the separate clonal contributions of two or more competing cell populations. To illustrate the power of this methodology, we demonstrate its ability to discriminate the metastatic behavior in immunodeficient mice of a well-established human metastatic cancer cell line and its co-transplanted  knockdown derivative. We also show how the use of machine learning to quantify clone-initiating cell (CIC) numbers and their subsequent metastatic progeny generated in different sites can reveal previously unknown relationships between different cellular genotypes and their initial sites of implantation with their subsequent respective dissemination patterns. These findings underscore the potential of such combined genomic and computational methodologies to identify new clonally-relevant drivers of site-specific patterns of metastasis.

DNA barcoded competitive clone-initiating cell analysis reveals novel features of metastatic growth in a cancer xenograft model.

T cells show tremendous efficacy as cellular therapeutics. However, obtaining primary T cells from human donors is expensive and variable. Pluripotent stem cells (PSCs) have the potential to provide a renewable source of T cells, but differentiating PSCs into hematopoietic progenitors with T cell potential remains an important challenge. Here, we report an efficient serum- and feeder-free system for differentiating human PSCs into hematopoietic progenitors and T cells. This fully defined approach allowed us to study the impact of individual proteins on blood emergence and differentiation. Providing DLL4 and VCAM1 during the endothelial-to-hematopoietic transition enhanced downstream progenitor T cell output by ~80-fold. These two proteins synergized to activate notch signaling in nascent hematopoietic stem and progenitor cells, and VCAM1 additionally promoted an inflammatory transcriptional program. We also established optimized medium formulations that enabled efficient and chemically defined maturation of functional CD8αβ, CD4, CD3, TCRαβ T cells with a diverse TCR repertoire.

Article	Year
Cord blood expansion. Pyrimidoindole derivatives are agonists of human hematopoietic stem cell self-renewal. Science (New York, N.Y.)\| PubMed ID: 25237102	2014
UM171 Enhances Lentiviral Gene Transfer and Recovery of Primitive Human Hematopoietic Cells. Molecular therapy. Methods & clinical development\| PubMed ID: 30101153	2018
Survey Says: "COVID-19 Lockdown Hits Young Faculty and Clinical Trials". Stem cell reports\| PubMed ID: 32574555	2020
Harnessing tRNA for Processing Ability and Promoter Activity. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 32926380	2021
A blood transcriptome-based analysis of disease progression, immune regulation, and symptoms in coronavirus-infected patients. Cell death discovery\| PubMed ID: 33293514	2020
DNA barcoded competitive clone-initiating cell analysis reveals novel features of metastatic growth in a cancer xenograft model. NAR cancer\| PubMed ID: 35875052	2022
DLL4 and VCAM1 enhance the emergence of T cell-competent hematopoietic progenitors from human pluripotent stem cells. Science advances\| PubMed ID: 36001668	2022

David J. H. F. Knapp

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