ex-vivo-expansion-genetic-manipulation-mouse-hematopoietic-stem-cells

<em>Ex Vivo</em> Expansion and Genetic Manipulation of Mouse Hematopoietic Stem Cells in Polyvinyl Alcohol-Based Cultures

Self-renewing&#160;multipotent&#160;hematopoietic stem cells (HSCs) are an important cell type due to their abilities to support hematopoiesis throughout ...

University of Oxford

The Mixed Lineage Leukemia (MLL) protein is an important epigenetic regulator required for the maintenance of gene activation during development. MLL chromosomal translocations produce novel fusion proteins that cause aggressive leukemias in humans. Individual MLL fusion proteins have distinct leukemic phenotypes even when expressed in the same cell type, but how this distinction is delineated on a molecular level is poorly understood. Here, we highlight a unique molecular mechanism whereby the RUNX1 gene is directly activated by MLL-AF4 and the RUNX1 protein interacts with the product of the reciprocal AF4-MLL translocation. These results support a mechanism of transformation whereby two oncogenic fusion proteins cooperate by activating a target gene and then modulating the function of its downstream product.

RUNX1 is a key target in t(4;11) leukemias that contributes to gene activation through an AF4-MLL complex interaction.

Transcription factors (TFs) act within wider regulatory networks to control cell identity and fate. Numerous TFs, including Scl (Tal1) and PU.1 (Spi1), are known regulators of developmental and adult haematopoiesis, but how they act within wider TF networks is still poorly understood. Transcription activator-like effectors (TALEs) are a novel class of genetic tool based on the modular DNA-binding domains of Xanthomonas TAL proteins, which enable DNA sequence-specific targeting and the manipulation of endogenous gene expression. Here, we report TALEs engineered to target the PU.1-14kb and Scl+40kb transcriptional enhancers as efficient new tools to perturb the expression of these key haematopoietic TFs. We confirmed the efficiency of these TALEs at the single-cell level using high-throughput RT-qPCR, which also allowed us to assess the consequences of both PU.1 activation and repression on wider TF networks during developmental haematopoiesis. Combined with comprehensive cellular assays, these experiments uncovered novel roles for PU.1 during early haematopoietic specification. Finally, transgenic mouse studies confirmed that the PU.1-14kb element is active at sites of definitive haematopoiesis in vivo and PU.1 is detectable in haemogenic endothelium and early committing blood cells. We therefore establish TALEs as powerful new tools to study the functionality of transcriptional networks that control developmental processes such as early haematopoiesis.

Single-cell analyses of regulatory network perturbations using enhancer-targeting TALEs suggest novel roles for PU.1 during haematopoietic specification.

The coordinated differentiation of hematopoietic stem and progenitor cells (HSPCs) into the various mature blood cell types is responsible for sustaining blood and immune system homeostasis. The cell fate decisions underlying this important biological process are made at the level of single cells. Methods to trace the fate of single cells are therefore essential for understanding hematopoietic system activity in health and disease and have had a major impact on how we understand and represent hematopoiesis. Here, we discuss the basic methodologies and technical considerations for three important clonal assays: single-cell transplantation, lentiviral barcoding, and Sleeping Beauty barcoding. This perspective is a synthesis of presentations and discussions from the 2019 International Society for Experimental Hematology (ISEH) Annual Meeting New Investigator Technology Session and the 2019 ISEH Winter Webinar.

Single-cell lineage tracing approaches in hematology research: technical considerations.

Bone marrow (BM) chimeric mice are a valuable tool in the field of immunology, with the genetic manipulation of donor cells widely used to study gene function under physiological and pathological settings. To date, however, BM chimera protocols require myeloablative conditioning of recipient mice, which dramatically alters steady-state hematopoiesis. Additionally, most protocols use fluorescence-activated cell sorting (FACS) of hematopoietic stem/progenitor cells (HSPCs) for ex vivo genetic manipulation. Here, we describe our development of cell culture techniques for the enrichment of functional HSPCs from mouse BM without the use of FACS purification. Furthermore, the large number of HSPCs derived from these cultures generate BM chimeric mice without irradiation. These HSPC cultures can also be genetically manipulated by viral transduction, to allow for doxycycline-inducible transgene expression in donor-derived immune cells within non-conditioned immunocompetent recipients. This technique is therefore expected to overcome current limitations in mouse transplantation models.

Non-conditioned bone marrow chimeric mouse generation using culture-based enrichment of hematopoietic stem and progenitor cells.

Ex vivo expansion of hematopoietic stem cells (HSCs) is one of the most promising strategies to increase the availability of transplantable HSCs and improve bone marrow transplantation outcomes. We recently demonstrated that mouse HSCs could be efficiently expanded in polyvinyl alcohol (PVA)-containing culture medium using only recombinant stem cell factor and thrombopoietin cytokines. However, the behavior of human HSCs in these simple PVA-based media was not fully elucidated. In this study, we analyzed the compatibility of PVA of different hydrolysis rates (HR) and molecular weights (MW) to support functional human and mouse HSCs ex vivo. Human and mouse HSCs proliferated more frequently in media containing PVA with lower HR than with higher HR, but both PVA types supported HSC multilineage reconstitution potential. Importantly, human HSCs cultured in PVA-containing media engrafted not only in irradiated recipients but also in non-irradiated recipients. Our results demonstrate that human HSCs can be maintained ex vivo using PVA-based culture systems and suggest approaches for future optimization of human HSC expansion.

Polyvinyl alcohol hydrolysis rate and molecular weight influence human and murine HSC activity ex vivo.

Bone marrow failure syndromes encompass a range of inherited and acquired hematological diseases that result in insufficient blood cell production, which leads to severe complications including anemia, weakening of the immune system, impaired coagulation, and increased risk of cancer. Within inherited bone marrow failure syndromes, a number of genetically distinct diseases have been described including Shwachman-Diamond syndrome and Fanconi anemia. Given the genetic complexity and poor prognosis of these inherited bone marrow failure syndromes, there is increasing interest in both characterizing the genetic landscapes of these diseases and developing novel gene therapies to effectively monitor and cure patients. These topics were the focus of the winter 2021 International Society for Experimental Hematology New Investigator Webinar, which featured presentations by Dr. Akiko Shimamura and Dr. Paula Río. Here, we review the topics covered within this webinar.

Translational research for bone marrow failure patients.

Hematopoietic stem cell transplantation (HSCT) is a curative therapy for a range of hematological diseases, from leukemias to immunodeficiencies and anemias. The aim in using HSCT is to replace a patient's dysfunctional blood system with a functional one by transplanting healthy hematopoietic stem cells (HSCs). HSCs may be collected from a healthy donor (for allogeneic HSCT) or from the patient for genetic correction (for autologous HSCT gene therapies). Despite the curative potential of HSCT, several hurdles to its wider and safer use remain, including how to efficiently genetically correct HSCs and how to increase donor HSC numbers to improve the donor pool. In recent years, the development of state-of-the-art technologies, such as Cas9-AAV6 technologies and identification of the small molecule HSC agonist UM171, have accelerated progress in HSC gene editing and expansion. These translational research efforts were the focus of the Spring 2021 International Society for Experimental Hematology (ISEH) webinar. Here we present a summary and discussion of the implications of these new approaches to improve HSC-based therapy.

Hematopoietic stem cell gene editing and expansion: State-of-the-art technologies and recent applications.

Cell and gene therapies using haematopoietic stem cells (HSCs) epitomize the transformative potential of regenerative medicine. Recent clinical successes for gene therapies involving autologous HSC transplantation (HSCT) demonstrate the potential of genetic engineering in this stem cell type for curing disease. With recent advances in CRISPR gene-editing technologies, methodologies for the ex vivo expansion of HSCs and non-genotoxic conditioning protocols, the range of clinical indications for HSC-based gene therapies is expected to significantly expand. However, substantial immunological challenges need to be overcome. These include pre-existing immunity to gene-therapy reagents, immune responses to neoantigens introduced into HSCs by genetic engineering, and unique challenges associated with next-generation and off-the-shelf HSC products. By synthesizing these factors in this Review, we hope to encourage more research to address the immunological issues associated with current and next-generation HSC-based gene therapies to help realize the full potential of this field.

Immunological barriers to haematopoietic stem cell gene therapy.

Hematopoietic stem cells (HSCs) cultured outside the body are the fundamental component of a wide range of cellular and gene therapies. Recent efforts have achieved > 200-fold expansion of functional HSCs, but their molecular characterization has not been possible since the majority of cells are non-HSCs and single cell-initiated cultures have substantial clone-to-clone variability. Using the Fgd5 reporter mouse in combination with the EPCR surface marker, we report exclusive identification of HSCs from non-HSCs in expansion cultures. By directly linking single-clone functional transplantation data with single-clone gene expression profiling, we show that the molecular profile of expanded HSCs is similar to proliferating fetal HSCs and reveals a gene expression signature, including Esam, Prdm16, Fstl1, and Palld, that can identify functional HSCs from multiple cellular states. This "repopulation signature" (RepopSig) also enriches for HSCs in human datasets. Together, these findings demonstrate the power of integrating functional and molecular datasets to better derive meaningful gene signatures and opens the opportunity for a wide range of functional screening and molecular experiments previously not possible due to limited HSC numbers.

Identification and characterization of in vitro expanded hematopoietic stem cells.

Lineage tracing using fluorescent proteins, genetic barcodes, and various other strategies have provided critical insights into the dynamics of both fetal and adult hematopoiesis in model organisms. However, these technologies cannot be readily utilized to study hematopoiesis in humans. Therefore, there is a critical need to develop strategies to assess cellular dynamics within human hematopoietic tissues in vivo. Recently, researchers have utilized naturally acquired somatic mutations, coupled with other single-cell technologies, to retrospectively analyze clonal cellular dynamics. In summer 2022 the International Society for Experimental Hematology's New Investigator Committee hosted a webinar focused on novel approaches to dissect fetal and adult hematopoiesis, with presentations from Drs. Ana Cvejic and Vijay Sankaran. Here, we provide an overview of these exciting technological advances and some of the novel insights they have already provided in studying human hematopoiesis.

Article	Year
RUNX1 is a key target in t(4;11) leukemias that contributes to gene activation through an AF4-MLL complex interaction. Cell reports\| PubMed ID: 23352661	2013
Single-cell analyses of regulatory network perturbations using enhancer-targeting TALEs suggest novel roles for PU.1 during haematopoietic specification. Development (Cambridge, England)\| PubMed ID: 25252941	2014
Single-cell lineage tracing approaches in hematology research: technical considerations. Experimental hematology\| PubMed ID: 32735908	2020
Non-conditioned bone marrow chimeric mouse generation using culture-based enrichment of hematopoietic stem and progenitor cells. Nature communications\| PubMed ID: 34117255	2021
Polyvinyl alcohol hydrolysis rate and molecular weight influence human and murine HSC activity ex vivo. Stem cell research\| PubMed ID: 34509158	2021
Translational research for bone marrow failure patients. Experimental hematology\| PubMed ID: 34801643	2022
Hematopoietic stem cell gene editing and expansion: State-of-the-art technologies and recent applications. Experimental hematology\| PubMed ID: 34973360	2022
Immunological barriers to haematopoietic stem cell gene therapy. Nature reviews. Immunology\| PubMed ID: 35301483	2022
Identification and characterization of in vitro expanded hematopoietic stem cells. EMBO reports\| PubMed ID: 35971894	2022
Exploiting somatic mutations to decipher human blood production: a natural lineage-tracing strategy. Experimental hematology\| PubMed ID: 36736573	2023

Adam C. Wilkinson

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