scholarly journals Screening of Chemically Distinct Lipid Nanoparticles In Vivo Using DNA Barcoding Technology Towards Effectively Delivering Messenger RNA to Hematopoietic Stem and Progenitor Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2931-2931
Author(s):  
Cory Sago ◽  
Elizabeth Campbell ◽  
Brianna Lutz ◽  
Neeraj Patwardhan ◽  
Gregory Hamilton ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Gene Editing ◽  
Ex Vivo ◽  
Cre Recombinase ◽  
Publicly Traded ◽  
Hematopoietic Stem ◽  
Cynomolgus Macaques ◽  
Stem And Progenitor Cells ◽  
Dose Dependent

Abstract Using adenine base editors, we aim to treat sickle cell disease by generating single nucleotide polymorphisms in human CD34+ hematopoietic stem and progenitor cells (HSPCs) at specific target sites by mediating A-T to G-C base conversions. While ex vivo gene editing approaches show great therapeutic promise, access is limited due to the requirement of an autologous hematopoietic stem cell (HSC) transplant to deliver the ex vivo edited cells. To further increase the number of patients eligible for base editing therapy, we are developing an alternative approach to directly deliver base editors to HSCs in vivo through non-viral delivery methods. Lipid Nanoparticles (LNPs) are a clinically validated, non-viral approach that enables the delivery of nucleic acid payloads, which may avoid the challenges associated with ex vivo approaches including the transplantation of edited CD34+ HSPCs. Here we describe the development and characterization of LNPs for the delivery of messenger RNA (mRNA) to HSPCs in vivo in both mice and cynomolgus macaques. By screening >1,000 chemically distinct LNPs in vivo utilizing a DNA barcoding technology, we identified several hit LNPs capable of biodistribution to HSPCs. Upon individual validation of these hit LNPs by delivery of Cre recombinase mRNA in a Cre-reporter mouse model (Ai14), which expresses the fluorescent protein tdTomato under a constitutive CAG promoter following Cre-meditate gene editing, we confirmed that several LNPs efficiently delivered Cre recombinase mRNA to mouse Lin-Sca-1+c-Kit+ (LSK) HSPCs. We next confirmed the most potent hit LNP (LNP-HSC1) identified from the in vivo screen to transfect LSK HSPCs in a dose-dependent manner between 0.1 and 1.0 mg/kg Cre recombinase mRNA, transfecting over 40% of LSK HSPCs in Ai14 mice at 1.0mg/kg. In a transfection durability study using Ai14 mice, we observed maintenance of tdTomato+ LSK HSPCs levels in the bone marrow at 10 weeks post-LNP delivery. As LNP-HSC1 had been identified and validated in mice of a C57BL6/j background, we next confirmed its ability to transfect a reporter mRNA into HSPCs in Balb/c mice and in 5 cynomolgus macaques. LNP-HSC1 efficiently transfected LSK HSPCs in Balb/c mice at doses ranging from 0.3 to 1.0 mg/kg. In 5 cynomolgus macaques (n=5 across two experiments), we observed a dose-dependent increase in reporter mRNA delivery with an average of 19% of bone marrow-derived CD34+ HSPCs (n=3) expressing the reporter protein at the highest dose tested. Taken together, these data demonstrate the value of our in vivo high-throughput LNP screening approach to identify novel LNPs capable of delivering to HSPCs, providing a promising delivery platform for an in vivo HSC gene editing approach for the treatment of hemoglobinopathies. Disclosures Sago: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Campbell: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lutz: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Patwardhan: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Hamilton: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Wong: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lee: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Keating: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Murray: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Singh: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ciaramella: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Efficient Nanoparticle-Mediated Delivery of Gene Editing Reagents into Human Hematopoietic Stem and Progenitor Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2933-2933
Author(s):  
Rkia El Kharrag ◽  
Kurt Berckmueller ◽  
Margaret Cui ◽  
Ravishankar Madhu ◽  
Anai M Perez ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Quality Control ◽  
Progenitor Cells ◽  
Gene Editing ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract Autologous hematopoietic stem cell (HSC) gene therapy has the potential to cure millions of patients suffering from hematological diseases and disorders. Recent HSCs gene therapy trials using CRISPR/Cas9 nucleases to treat sickle cell disease (SCD) have shown promising results paving the way for gene editing approaches for other diseases. However, current applications depend on expensive and rare GMP facilities for the manipulation of HSCs ex vivo. Consequently, this promising treatment option remains inaccessible to many patients especially in low- and middle-income settings. HSC-targeted in vivo delivery of gene therapy reagents could overcome this bottleneck and thereby enhance the portability and availability of gene therapy. Various kinds of nanoparticles (lipid, gold, polymer, etc.) are currently used to develop targeted ex vivo as well as in vivo gene therapy approaches. We have previously shown that poly (β-amino ester) (PBAE)-based nanoparticle (NP) formulations can be used to efficiently deliver mRNA into human T cells and umbilical cord blood-derived CD34 + hematopoietic stem and progenitor cells (HSPCs) (Moffet et al. 2017, Nature Communications). Here, we optimized our NP formulation to deliver mRNA into GCSF-mobilized adult human CD34 + HSPCs, a more clinically relevant and frequently used cell source for ex vivo and the primary target for in vivo gene therapy. Furthermore, we specifically focused on the evaluation of NP-mediated delivery of CRISPR/Cas9 gene editing reagents. The efficiency of our NP-mediated delivery of gene editing reagents was comprehensively tested in comparison to electroporation, the current experimental, pre-clinical as well as clinical standard for gene editing. Most important for the clinical translation of this technology, we defined quality control parameters for NPs, identified standards that can predict the editing efficiency, and established protocols to lyophilize and store formulated NPs for enhanced portability and future in vivo applications. Nanoformulations were loaded with Cas9 ribonucleoprotein (RNP) complexes to knock out CD33, an established strategy in our lab to protect HSCs from anti-CD33 targeted acute myeloid leukemia (AML) immunotherapy (Humbert et al. 2019, Leukemia). RNP-loaded NPs were evaluated for size and charge to correlate physiochemical properties with the outcome as well as establish quality control standards. NPs passing the QC were incubated with human GCSF-mobilized CD34 + hematopoietic stem and progenitor cells (HSPCs). In parallel, RNPs were delivered into CD34 + cells using our established EP protocol. NP- and EP-edited CD34 + cells were evaluated phenotypically by flow cytometry and functionally in colony-forming cell (CFC) assays as well as in NSG xenograft model. The optimal characteristics for RNP-loaded NPs were determined at 150-250 nm and 25-35 mV. Physiochemical assessment of RNP-loaded NP formations provided an upfront quality control of RNP components reliably detecting degraded components. Most importantly, NP charge directly correlated with the editing efficiency (Figure A). NPs achieved more than 85% CD33 knockout using 3-fold lower dose of CRISPR nucleases compared to EP. No impact on the erythromyeloid differentiation potential of gene-edited cells in CFC assays was observed. Finally, NP-modified CD34 + cells showed efficient and sustained gene editing in vivo with improved long-term multilineage engraftment potential in the peripheral blood (PB) and bone marrow stem cell compartment of NSG mice in comparison to EP-edited cells (Figure B). Here we show that PBAE-NPs enable efficient CRISPR/Cas9 gene editing of human GCSF-mobilized CD34 + cells without compromising the viability and long-term multilineage engraftment of human HSPCs in vivo. Most importantly, we defined physiochemical properties of PBAE-NPs that enable us to not only determine the integrity of our gene-editing agents but also predict the efficiency of editing in HSPCs. The requirement of 3-fold less reagents compared to EP, the ability to lyophilize quality-controlled and ready to administer gene therapy reagents, and the opportunity to engineer the surface of PBAE-NPs with HSC-targeting molecules (e.g. antibodies) could make this also a highly attractive and portable editing platform for in vivo HSC gene therapy. Figure 1 Figure 1. Disclosures Kiem: VOR Biopharma: Consultancy; Homology Medicines: Consultancy; Ensoma Inc.: Consultancy, Current holder of individual stocks in a privately-held company. Radtke: Ensoma Inc.: Consultancy; 47 Inc.: Consultancy.

Organ-Selective Homing Defines Engraftment Kinetics of Murine Hematopoietic Stem Cells and Is Compromised by Ex Vivo Expansion

Blood ◽  
1999 ◽  
Vol 93 (5) ◽  
pp. 1557-1566 ◽  
Author(s):  
Stephen J. Szilvassy ◽  
Michael J. Bass ◽  
Gary Van Zant ◽  
Barry Grimes
Keyword(s):  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Hematopoietic Reconstitution ◽  
Dramatic Decline ◽  
Stem And Progenitor Cells ◽  
Clonogenic Cells

Abstract Hematopoietic reconstitution of ablated recipients requires that intravenously (IV) transplanted stem and progenitor cells “home” to organs that support their proliferation and differentiation. To examine the possible relationship between homing properties and subsequent engraftment potential, murine bone marrow (BM) cells were labeled with fluorescent PKH26 dye and injected into lethally irradiated hosts. PKH26+ cells homing to marrow or spleen were then isolated by fluorescence-activated cell sorting and assayed for in vitro colony-forming cells (CFCs). Progenitors accumulated rapidly in the spleen, but declined to only 6% of input numbers after 24 hours. Although egress from this organ was accompanied by a simultaneous accumulation of CFCs in the BM (plateauing at 6% to 8% of input after 3 hours), spleen cells remained enriched in donor CFCs compared with marrow during this time. To determine whether this differential homing of clonogenic cells to the marrow and spleen influenced their contribution to short-term or long-term hematopoiesis in vivo, PKH26+ cells were sorted from each organ 3 hours after transplantation and injected into lethally irradiated Ly-5 congenic mice. Cells that had homed initially to the spleen regenerated circulating leukocytes (20% of normal counts) approximately 2 weeks faster than cells that had homed to the marrow, or PKH26-labeled cells that had not been selected by a prior homing step. Both primary (17 weeks) and secondary (10 weeks) recipients of “spleen-homed” cells also contained approximately 50% higher numbers of CFCs per femur than recipients of “BM-homed” cells. To examine whether progenitor homing was altered upon ex vivo expansion, highly enriched Sca-1+c-kit+Lin−cells were cultured for 9 days in serum-free medium containing interleukin (IL)-6, IL-11, granulocyte colony-stimulating factor, stem cell factor, flk-2/flt3 ligand, and thrombopoietin. Expanded cells were then stained with PKH26 and assayed as above. Strikingly, CFCs generated in vitro exhibited a 10-fold reduction in homing capacity compared with fresh progenitors. These studies demonstrate that clonogenic cells with differential homing properties contribute variably to early and late hematopoiesis in vivo. The dramatic decline in the homing capacity of progenitors generated in vitro underscores critical qualitative changes that may compromise their biologic function and potential clinical utility, despite their efficient numerical expansion.

scholarly journals Extracellular vesicles of stromal origin target and support hematopoietic stem and progenitor cells

2017 ◽  
Vol 216 (7) ◽  
pp. 2217-2230 ◽  
Author(s):  
Gregoire Stik ◽  
Simon Crequit ◽  
Laurence Petit ◽  
Jennifer Durant ◽  
Pierre Charbord ◽  
...  
Keyword(s):  
Gene Expression ◽  
Progenitor Cells ◽  
Extracellular Vesicles ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Critical Role ◽  
Molecular Signature ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Extracellular vesicles (EVs) have been recently reported as crucial mediators in cell-to-cell communication in development and disease. In this study, we investigate whether mesenchymal stromal cells that constitute a supportive microenvironment for hematopoietic stem and progenitor cells (HSPCs) released EVs that could affect the gene expression and function of HSPCs. By taking advantage of two fetal liver–derived stromal lines with widely differing abilities to maintain HSPCs ex vivo, we demonstrate that stromal EVs play a critical role in the regulation of HSPCs. Both supportive and nonsupportive stromal lines secreted EVs, but only those delivered by the supportive line were taken up by HSPCs ex vivo and in vivo. These EVs harbored a specific molecular signature, modulated the gene expression in HSPCs after uptake, and maintained the survival and clonogenic potential of HSPCs, presumably by preventing apoptosis. In conclusion, our study reveals that EVs are an important component of the HSPC niche, which may have major applications in regenerative medicine.

scholarly journals CRISPR Therapeutics: New Emerging Developments and Clinical Applications

2021 ◽  
Vol 11 (5) ◽  
pp. 193-195
Author(s):  
Kaiser Jay Aziz-Andersen
Keyword(s):  
Stem Cells ◽  
Gene Editing ◽  
Ex Vivo ◽  
Human Subjects ◽  
Clinical Applications ◽  
Approval Process ◽  
Hematopoietic Stem ◽  
Immune System Diseases ◽  
Wide Range

CRISPR gene editing is a genetic engineering technique applied in clinical applications in which the genomes of living organisms may be modified. It is based on the principles of the CRISPR-Cas9 antiviral defense system. It is based on delivering the Cas9 nuclease complexed with a synthetic guide RNA into a living organism cell and that organisms’s genome can be “cut” and –“paste” at a desired location, allowing existing genes to be modified for desired outcome (i.e., CRISPR for Precision Medicine). CRISPR gene editing harnesses the natural defense mechanisms of some bacteria to cut human DNA strands. Then the DNA strand either heals itself or injects a new piece of DNA to mend the gap. Studies have been reported in Lung Cancer diagnosis and treatments. CRISPR-based engineering techniques have been developed for T Cells and Stem cells applications (i.e. Gene Corrections in Hematopoietic Stem Cells for the Treatment of Blood and Immune System Diseases). Even though earlier CRISPR methodologies were used for performing simple DNA edits, recent applications include the ability to delete genes or insert genes, and edit regulatory regions in a wide range of cell types. The role of CRISPR in human therapeutics is currently focused on utilizing CRISPR techniques to perform either in vivo editing of human cells–everything from the head, eye all the way to neurons and liver cells--or performing ex vivo therapies. The FDA’s new genomic CRISPR technology based products approval process begins with review and evaluation of preclinical studies in order to establish and characterize the proposed product’s safety profile. New genomic products must be shown to be safe and effective for the FDA approval process. The sponsor of the new genomic product must show that the product is safe and effective in human subjects.1

scholarly journals Mitochondrial augmentation of CD34+ cells from healthy donors and patients with mitochondrial DNA disorders confers functional benefit

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Elad Jacoby ◽  
Moriya Ben Yakir-Blumkin ◽  
Shiri Blumenfeld-Kan ◽  
Yehuda Brody ◽  
Amilia Meir ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Mouse Model ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Functional Benefit ◽  
Syngeneic Mouse Model ◽  
Multisystemic Disease ◽  
Stem And Progenitor Cells ◽  
Disease Modifying

AbstractMitochondria are cellular organelles critical for numerous cellular processes and harboring their own circular mitochondrial DNA (mtDNA). Most mtDNA associated disorders (either deletions, mutations, or depletion) lead to multisystemic disease, often severe at a young age, with no disease-modifying therapies. Mitochondria have a capacity to enter eukaryotic cells and to be transported between cells. We describe a method of ex vivo augmentation of hematopoietic stem and progenitor cells (HSPCs) with normal exogenous mitochondria, termed mitochondrial augmentation therapy (MAT). Here, we show that MAT is feasible and dose dependent, and improves mitochondrial content and oxygen consumption of healthy and diseased HSPCs. Ex vivo mitochondrial augmentation of HSPCs from a patient with a mtDNA disorder leads to superior human engraftment in a non-conditioned NSGS mouse model. Using a syngeneic mouse model of accumulating mitochondrial dysfunction (Polg), we show durable engraftment in non-conditioned animals, with in vivo transfer of mitochondria to recipient hematopoietic cells. Taken together, this study supports MAT as a potential disease-modifying therapy for mtDNA disorders.

scholarly journals EnhancedEx VivoExpansion of Human Hematopoietic Progenitors on Native and Spin Coated Acellular Matrices Prepared from Bone Marrow Stromal Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-13 ◽  
Author(s):  
Samiksha Wasnik ◽  
Suma Kantipudi ◽  
Mark A. Kirkland ◽  
Gopal Pande
Keyword(s):  
Bone Marrow ◽  
Ex Vivo ◽  
Native Form ◽  
Hematopoietic Stem ◽  
Lineage Specific Expansion ◽  
Stem And Progenitor Cells ◽  
Stromal Cell Line ◽  
Specific Expansion ◽  
Efficient Expansion

The extracellular microenvironment in bone marrow (BM) is known to regulate the growth and differentiation of hematopoietic stem and progenitor cells (HSPC). We have developed cell-free matrices from a BM stromal cell line (HS-5), which can be used as substrates either in native form or as tissue engineered coatings, for the enhancedex vivoexpansion of umbilical cord blood (UCB) derived HSPC. The physicochemical properties (surface roughness, thickness, and uniformity) of native and spin coated acellular matrices (ACM) were studied using scanning and atomic force microscopy (SEM and AFM). Lineage-specific expansion of HSPC, grown on these substrates, was evaluated by immunophenotypic (flow cytometry) and functional (colony forming) assays. Our results show that the most efficient expansion of lineage-specific HSPC occurred on spin coated ACM. Our method provides an improved protocol forex vivoHSPC expansion and it offers a system to study thein vivoroles of specific molecules in the hematopoietic niche that influence HSPC expansion.

scholarly journals Advances and Obstacles in Homology-Mediated Gene Editing of Hematopoietic Stem Cells

2021 ◽  
Vol 10 (3) ◽  
pp. 513
Author(s):  
Christi T. Salisbury-Ruf ◽  
Andre Larochelle
Keyword(s):  
Gene Editing ◽  
High Efficiency ◽  
Viral Vector ◽  
Ex Vivo ◽  
Blood Disorders ◽  
Hematopoietic Stem ◽  
Gene Therapies ◽  
Clinical Investigations ◽  
Stem And Progenitor Cells ◽  
Programmable Nucleases

Homology-directed gene editing of hematopoietic stem and progenitor cells (HSPCs) is a promising strategy for the treatment of inherited blood disorders, obviating many of the limitations associated with viral vector-mediated gene therapies. The use of CRISPR/Cas9 or other programmable nucleases and improved methods of homology template delivery have enabled precise ex vivo gene editing. These transformative advances have also highlighted technical challenges to achieve high-efficiency gene editing in HSPCs for therapeutic applications. In this review, we discuss recent pre-clinical investigations utilizing homology-mediated gene editing in HSPCs and highlight various strategies to improve editing efficiency in these cells.

scholarly journals Selective Targeting of Splicing Factor Mutant Hematopoietic Stem and Progenitor Cells Via STAT3 Inhibition

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1509-1509
Author(s):  
Kathryn S Potts ◽  
Rosannah C. Cameron ◽  
Noura Ghazale ◽  
Varun Gupta ◽  
Juan Martin Barajas ◽  
...  
Keyword(s):  
Research Funding ◽  
Synthetic Lethality ◽  
K562 Cells ◽  
Splicing Factor ◽  
Publicly Traded ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Mutant Cells ◽  
Stat3 Inhibition

Abstract Myelodysplastic syndrome (MDS) is a bone marrow failure disorder driven by dysfunction of hematopoietic stem and progenitor cells (HSPCs). Patient sequencing studies over the last decade have revealed that mutations in splicing machinery predominate in MDS, thus selective targeting of these cells is therapeutically attractive. STAT3 inhibition has been explored previously as a means to eradicate HSPCs in MDS. While efficacy was demonstrated in a subset of samples, the underlying mechanism for this selectivity remains unknown. We examined RNAseq of MDS CD34+ HSPCs with splicing factor mutations versus wildtype, finding alternative splicing and differential expression of STAT3 pathway components. Functionally, we explored if STAT3 signaling represents a novel vulnerability in SF3B1 mutant HSPCs using a multi-model approach of in vivo zebrafish and mouse systems, and in vitro assays of CRISPR-engineered human leukemia K562 cells and primary MDS samples. Utilizing the small molecule STAT3 inhibitor STATTIC, we found that human cells carrying MDS-associated SF3B1 point mutations had heightened sensitivity to STAT3 inhibition compared to wildtype controls. To evaluate the activity of STAT3 inhibition in vivo, we utilized an Mx1-cre conditional knock-in mouse model of mutant SF3B1 (Sf3b1+/K700E). We demonstrated that in vivo STATTIC treatment selectively depleted Sf3b1 mutant cells over wildtype in vivo. RNAseq of sf3b1 homozygous mutantzebrafish cells revealed conserved dysregulation of STAT3 pathway splicing and target expression. Diminishing Stat3 (via morpholino knockdown, stable mutants, or STATTIC treatment) decreased HSPCs in sf3b1 heterozygotes but not wildtype embryos, demonstrating synthetic lethality between Sf3b1 and Stat3. Our data indicate that SF3B1 heterozygosity, regardless of the type of mutation, confers a heightened sensitivity to STAT3 inhibition in zebrafish, mouse, and human HSPCs. Critically, our data indicate that SF3B1-mutant cells can be selectively killed in vivo while sparing wildtype cells. We sought to rescue HSPCs in sf3b1 homozygous mutant zebrafish, however overexpression of ligands Osm and Il6 or wildtype Stat3 was insufficient. Instead, overexpression of constitutively-active Stat3 partially restored HSPCs, indicating that functional Stat3 signaling downstream of Sf3b1 is critical for HSPC formation. To investigate the specificity of the synthetic lethality for SF3B1, we assessed the STAT3 synthetic lethal interaction with other mutated splicing factors in MDS. Similar to SF3B1, we demonstrated STAT3 synthetic lethality with U2AF1 and SRSF2 heterozygosity in zebrafish and human cells. RNA-sequencing analysis of STATTIC-treated K562 cells revealed an exacerbation of splicing alterations upon STAT3 inhibition that was more pronounced in SF3B1+/K666N cells compared to wildtype. Even more strikingly, we demonstrated that constitutive activation of STAT3 could partially reverse defective splicing in zebrafish sf3b1 homozygous mutant cells. Mechanistically, these data strongly support coordinated splicing dysfunction as the underlying cause for STAT3-SF3B1 synthetic lethality. Together, we demonstrated a conserved and selective synthetic lethal interaction between STAT3 function and splicing factor defects that represents a novel liability for mutant HSPCs with important implications for MDS treatment. Disclosures Shastri: Guidepoint: Consultancy; Kymera Therapeutics: Research Funding; Onclive: Honoraria; GLC: Consultancy. Verma: Curis: Research Funding; BMS: Research Funding; Stelexis: Consultancy, Current equity holder in publicly-traded company; Eli Lilly: Research Funding; Medpacto: Research Funding; Incyte: Research Funding; GSK: Research Funding; Novartis: Consultancy; Acceleron: Consultancy; Celgene: Consultancy; Stelexis: Current equity holder in publicly-traded company; Throws Exception: Current equity holder in publicly-traded company.

Organ-Selective Homing Defines Engraftment Kinetics of Murine Hematopoietic Stem Cells and Is Compromised by Ex Vivo Expansion

Blood ◽  
1999 ◽  
Vol 93 (5) ◽  
pp. 1557-1566 ◽  
Author(s):  
Stephen J. Szilvassy ◽  
Michael J. Bass ◽  
Gary Van Zant ◽  
Barry Grimes
Keyword(s):  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Hematopoietic Reconstitution ◽  
Dramatic Decline ◽  
Stem And Progenitor Cells ◽  
Clonogenic Cells

Hematopoietic reconstitution of ablated recipients requires that intravenously (IV) transplanted stem and progenitor cells “home” to organs that support their proliferation and differentiation. To examine the possible relationship between homing properties and subsequent engraftment potential, murine bone marrow (BM) cells were labeled with fluorescent PKH26 dye and injected into lethally irradiated hosts. PKH26+ cells homing to marrow or spleen were then isolated by fluorescence-activated cell sorting and assayed for in vitro colony-forming cells (CFCs). Progenitors accumulated rapidly in the spleen, but declined to only 6% of input numbers after 24 hours. Although egress from this organ was accompanied by a simultaneous accumulation of CFCs in the BM (plateauing at 6% to 8% of input after 3 hours), spleen cells remained enriched in donor CFCs compared with marrow during this time. To determine whether this differential homing of clonogenic cells to the marrow and spleen influenced their contribution to short-term or long-term hematopoiesis in vivo, PKH26+ cells were sorted from each organ 3 hours after transplantation and injected into lethally irradiated Ly-5 congenic mice. Cells that had homed initially to the spleen regenerated circulating leukocytes (20% of normal counts) approximately 2 weeks faster than cells that had homed to the marrow, or PKH26-labeled cells that had not been selected by a prior homing step. Both primary (17 weeks) and secondary (10 weeks) recipients of “spleen-homed” cells also contained approximately 50% higher numbers of CFCs per femur than recipients of “BM-homed” cells. To examine whether progenitor homing was altered upon ex vivo expansion, highly enriched Sca-1+c-kit+Lin−cells were cultured for 9 days in serum-free medium containing interleukin (IL)-6, IL-11, granulocyte colony-stimulating factor, stem cell factor, flk-2/flt3 ligand, and thrombopoietin. Expanded cells were then stained with PKH26 and assayed as above. Strikingly, CFCs generated in vitro exhibited a 10-fold reduction in homing capacity compared with fresh progenitors. These studies demonstrate that clonogenic cells with differential homing properties contribute variably to early and late hematopoiesis in vivo. The dramatic decline in the homing capacity of progenitors generated in vitro underscores critical qualitative changes that may compromise their biologic function and potential clinical utility, despite their efficient numerical expansion.

Expansion of functionally defined mouse hematopoietic stem and progenitor cells by a short isoform of RUNX1/AML1

Blood ◽  
2012 ◽  
Vol 119 (3) ◽  
pp. 727-735 ◽  
Author(s):  
Shinobu Tsuzuki ◽  
Masao Seto
Keyword(s):  
Progenitor Cell ◽  
Gene Expression Analysis ◽  
Ex Vivo ◽  
Transcriptional Regulators ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Limited Success ◽  
Stem And Progenitor Cells

Abstract Self-renewal activity is essential for the maintenance and regeneration of the hematopoietic system. The search for molecules capable of promoting self-renewal and expanding hematopoietic stem cells (HSCs) has met with limited success. Here, we show that a short isoform (AML1a) of RUNX1/AML1 has such activities. Enforced AML1a expression expanded functionally defined HSCs, with an efficiency that was at least 20 times greater than that of the control in vivo and by 18-fold within 7 days ex vivo. The ex vivo–expanded HSCs could repopulate hosts after secondary transplantations. Moreover, AML1a expression resulted in vigorous and long-term (> 106-fold at 4 weeks) ex vivo expansion of progenitor cell populations capable of differentiating into multilineages. Gene expression analysis revealed that AML1a expression was associated with up-regulation of genes, including Hoxa9, Meis1, Stat1, and Ski. shRNA-mediated silencing of these genes attenuated AML1a-mediated activities. Overall, these findings establish AML1a as an isoform-specific molecule that can influence several transcriptional regulators associated with HSCs, leading to enhanced self-renewal activity and hematopoietic stem/progenitor cell expansion ex vivo and in vivo. Therefore, the abilities of AML1a may have implications for HSC transplantation and transfusion medicine, given that the effects also can be obtained by cell-penetrating AML1a protein.

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