Human Human Hematopoietic Stem Cells Research Articles

In vitro generation of epidermal keratinocytes from human CD34-positive hematopoietic stem cells.

The epidermis is largely composed of keratinocytes (KCs), and the proliferation and differentiation of KCs from the stratum basale to the stratum corneum is the cellular hierarchy present in the epidermis. In this study, we explore the differentiation abilities of human hematopoietic stem cells (HSCs) into KCs. Cultured HSCs positive for CD34, CD45, and CD133 with prominent telomerase activity were induced with keratinocyte differentiation medium (KDM), which is composed of bovine pituitary extract (BPE), epidermal growth factor (EGF), insulin, hydrocortisone, epinephrine, transferrin, calcium chloride (CaCl2), bone morphogenetic protein 4 (BMP4), and retinoic acid (RA). Differentiation was monitored through the expression of cytokeratin markers K5 (keratin 5), K14 (keratin 14), K10 (keratin 10), K1 (keratin 1), transglutaminase 1 (TGM1), involucrin (IVL), and filaggrin (FLG) on day 0 (D0), day 6 (D6), day 11 (D11), day 18 (D18), day 24 (D24), and day 30 (D30) using immunocytochemistry, fluorescence microscopy, flow cytometry, qPCR, and Western blotting. The results revealed the expression of K5 and K14 genes in D6 cells (early keratinocytes), K10 and K1 genes in D11-D18 cells (mature keratinocytes) with active telomerase enzyme, and FLG, IVL, and TGM1 in D18-D24 cells (terminal keratinocytes), and by D30, the KCs were completely enucleated similar to cornified matrix. This method of differentiation of HSCs to KCs explains the cellular order exists in the normal epidermis and opens the possibility of exploring the use of human HSCs in the epidermal differentiation.

Abstract PR017: Properties of somatic methylation changes in human hematopoietic stem cells in health and disease

Abstract Cancer is the proliferation of a genetic clone at the expense of its neighbours and normal tissue function. Studies have shown that with ageing, normal tissues become colonised by expanding clones, which acquired fitness-enhancing mutations that allow them to over-proliferate relative to their neighbours. However, whether the clones’ founding cell also carries an epigenome that is inherited by the expanding clone is incompletely understood. In the following study, we performed multi-omic profiling of human hematopoietic stem cells (HSCs) to establish the role of epigenetic heritability through time in health and disease. We sequenced whole genomes and methylomes of single human HSCs from three healthy individuals and several individuals diagnosed with myeloproliferative neoplasm or chronic myeloid leukaemia. Based on the patterns of shared and sample-specific somatic mutations, we built phylogenies of HSCs for each individual. To establish whether loss or gain of methylation, both partial and complete, was heritable, we developed a method which analyses read counts at a CpG site across cells together with their phylogeny. Our model assumes CpG sites are in one of three states (i.e. they are 0%, 50% or 100% methylated, as measured by the number of methylated reads divided by number of total reads at a site), methylation states are inherited during cell division and a cell’s state can spontaneously transition to any of the other two. Our method finds sites, which follow these assumptions, and proposes the most likely zygotic starting state and identifies phylogeny’s branches, which underwent a change, to produce the observed methylation profile of cells at a CpG site. We analysed approximately 25 million CpG sites per individual, and we find that the majority of sites exhibit heritability of methylation states with the aforementioned properties. The method allows us to accurately time when methylation changes occurred as the branches of a phylogeny can be linked to a developmental period, thus disentangling their evolutionary history. Applying the method across millions of CpG sites and thousands of single cells from these individuals, we found the rates of methylation change is several folds higher than the rate of somatic mutations and that methylation changes are stochastic and allele-specific. Moreover, we observe that these changes are remarkably stable as we found that hundreds of such changes are acquired between the time of X-chromosome inactivation and germ layer formation and are stably inherited until old age. In addition, we find that some of these changes precede the neoplasm’s clone, which suggests they might play a role in function. By comparing normal healthy individuals of different ages with cancer patients, we are able to unravel the process of normal ageing from disease development. Citation Format: Lori D. Kregar, Nicholas Williams, Joe Lee, Jyoti Nangalia, Peter Campbell. Properties of somatic methylation changes in human hematopoietic stem cells in health and disease [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr PR017.

Humanized mouse model for vaccine evaluation: an overview.

Animal models are essential in medical research for testing drugs and vaccines. These models differ from humans in various respects, so their results are not directly translatable in humans. To address this issue, humanized mice engrafted with functional human cells or tissue can be helpful. We propose using humanized mice that support the engraftment of human hematopoietic stem cells (HSCs) without irradiation to evaluate vaccines that influence patient immunity. For infectious diseases, several types of antigens and adjuvants have been developed and evaluated for vaccination. Peptide vaccines are generally used for their capability to fight cancer and infectious diseases. Evaluation of adjuvants is necessary as they induce inflammation, which is effective for an enhanced immune response but causes adverse effects in some individuals. A trial can be done on humanized mice to check the immunogenicity of a particular adjuvant and peptide combination. Messenger RNA has also emerged as a potential vaccine against viruses. These vaccines need to be tested with human immune cells because they work by producing a particular peptide of the pathogen. Humanized mice with human HSCs that can produce both myeloid and lymphoid cells show a similar immune response that these vaccines will produce in a patient.

Purging myeloma cell contaminants and simultaneous expansion of peripheral blood-mobilized stem cells

Human hematopoietic stem cells (HSCs) are widely used as a cellular source for hematopoietic stem cell transplantation (HSCT) in the clinical treatment of hematological malignancies. After transplantation therapy, delays in hematopoietic recovery due to insufficient donor-derived HSCs can lead to increased risks of life-threatening infections and bleeding. Our previous studies developed an efficient ex vivo expansion culture medium (3a medium) for umbilical cord blood-derived HSCs (CBSCs), offering a potential solution to this problem. Nevertheless, the broader applicability of our culture method to alternative cell sources and, of greater significance, its efficacy in eliminating potentially disease-associated contaminated tumor cells, especially in autologous transplantation, raise critical clinical questions. In this study, we modified the 3a medium by incorporating UM729 to replace UM171, adding FMS-like tyrosine kinase 3 (Flt3) ligand, and adjusting the concentrations of butyzamide, 740Y-P, polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (PCL-PVAc-PEG, Soluplus) to create the modified-3a medium. This sophistication allowed the efficient expansion of not only CBSCs but also peripheral blood-mobilized HSCs (PBSCs). Additionally, we successfully removed contaminated myeloma cells by adding bortezomib and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) at appropriate concentrations, although we maintained HSCs through the addition of lenalidomide. Our research findings present the potential for widespread clinical application of the modified-3a medium and suggest a safe ex vivo culture technique for expanding human HSCs within peripheral blood-derived donor grafts used for autologous HSCT.

Single Cell Analysis Identifies Expression of PD-L2 on Human Bone Marrow Hematopoietic Stem Cells

The identification of specific and reliable markers for human hematopoietic stem cells (HSCs) is crucial to enable their prospective isolation allowing the investigation of unique physiological HSC functions within the blood cell hierarchy. Despite significant advancements in identification of surface markers for human bone marrow HSC isolation, the enriched populations remain heterogeneous. Here we applied single cell CITE-Seq (cellular indexing of transcriptomes and epitopes) of FACS-enriched hematopoietic stem and progenitor cells (HSPCs) to decipher differentiation hierarchies and to identify markers of most immature populations. We FACS-isolated over 60.000 high-quality human bone marrow HSPCs by sorting CD34 + and CD34 +CD38 - cells from 15 different healthy donors. These were subjected to scCITE-Seq using the BD Rhapsody system with a targeted gene panel comprising 600 most relevant genes of early HSC differentiation and 50 antibodies. To validate differentially expressed surface marker genes, we performed extensive in vitro and in vivo studies of FACS-purified subpopulations of human bone marrow and mobilized peripheral blood samples (mPB) from healthy donors. Our multi-omics based single HSPC profiling indicated Programmed death ligand 2 (PD-L2 / CD273) as a surface marker expressed on the most immature HSCs within the hematopoietic compartment defined as CD34 +CD38 -CD45RA -CD90 +. Prospective FACS-based isolation allowed a deeper understanding of transcriptomic differences depending on PD-L2 positivity. A transcriptomic principal component analysis of CD273 hi and CD273 low expressing HSCs isolated pairwise from four healthy donors proved distinct clustering accompanied by discriminative molecular signatures. Stem cell genes as HOPX, MLLT3 and MPL were enriched in CD273 hi HSCs, and CDK6 was downregulated, further validating our single-cell analysis. The differential expression of stem cell genes was validated at protein level. Single cell tracking using video-microscopy confirmed a delayed entry into cell cycle in CD273 hi HSCs, suggesting deeper quiescence of these cells. Furthermore, their mitochondrial potential was reduced. In vivo transplantations of both populations in immunocompromised NSGs are ongoing and will reveal the long-term repopulation potential of CD273 hi HSCs. Multiple in vitro differentiation assays demonstrated CD273 positivity to be associated with an increased stem cell phenotype with higher expression of EPCR (CD201) and CD90 in liquid culture, a delayed differentiation and high capability of generating multipotent colonies. Interestingly, CD273 surface expression was strongly upregulated in HSCs upon in vitro culture. Since PD-L2 is known as a ligand for the Programmed death receptor 1 (PD1), we investigated the immunomodulating function of PD-L2 expressing HSPCs by establishing an allogeneic lymphocyte reaction assay. In addition to their stem cell-like characteristics, PD-L2 expressing HSPCs mediated diminished T cell proliferation in allogeneic co-culture settings. Furthermore, cytokine profiling of co-culture supernatants indicated increased secretion of anti-inflammatory cytokines (IL-10 and TGF-β1), accompanied by reduced secretion of pro-inflammatory cytokines (IL-17A) and a diminished expression of the pro-inflammatory receptor CD69 on CD8 + T cells. These findings showed that CD34 + cells isolated from mobilized peripheral blood of healthy donors possess immunomodulating capabilities, and that PD-L2 expression indicates an immunocompromising HSC phenotype. In conclusion, this study demonstrates differential surface expression of PD-L2 within the most immature human HSCs. PD-L2 expression is associated with HSC quiescence and delayed in vitro differentiation. Furthermore, these results provide valuable insights into the immunomodulating features of HSCs expressing PD-L2.

Targeted, safe, and efficient gene delivery to human hematopoietic stem and progenitor cells in vivo using the engineered AVID adenovirus vector platform

Targeted delivery and cell-type-specific expression of gene-editing proteins in various cell types invivo represent major challenges for all viral and non-viral delivery platforms developed to date. Here, we describe the development and analysis of artificial vectors for intravascular delivery (AVIDs), an engineered adenovirus-based gene delivery platform that allows for highly targeted, safe, and efficient gene delivery to human hematopoietic stem and progenitor cells (HSPCs) invivo after intravenous vector administration. Due to a set of refined structural modifications, intravenous administration of AVIDs did not trigger cytokine storm, hepatotoxicity, or thrombocytopenia. Single intravenous administration of AVIDs to humanized mice, grafted with human CD34+ cells, led to up to 20% transduction of CD34+CD38-CD45RA- HSPC subsets in the bone marrow. Importantly, targeted invivo transduction of CD34+CD38-CD45RA-CD90-CD49f+ subsets, highly enriched for human hematopoietic stem cells (HSCs), reached up to 19%, which represented a 1,900-fold selectivity in gene delivery to HSC-enriched over lineage-committed CD34-negative cell populations. Because the AVID platform allows for regulated, cell-type-specific expression of gene-editing technologies as well as expression of immunomodulatory proteins to ensure persistence of corrected HSCs invivo, the HSC-targeted AVID platform may enable development of curative therapies through invivo gene correction in human HSCs after a single intravenous administration.

Nuclear Tubulin Enhances CXCR4 Transcription and Promotes Chemotaxis Through TCF12 Transcription Factor in human Hematopoietic Stem Cells.

Tubulins are cytoskeleton components in all eukaryotic cells and play crucial roles in various cellular activities by polymerizing into dynamic microtubules. A subpopulation of tubulin has been shown to localize in the nucleus, however, the function of nuclear tubulin remains largely unexplored. Here we report that microtubule depolymerization specifically upregulates surface CXCR4 expression in human hematopoietic stem cells (HSCs). Mechanistically, microtubule depolymerization results in accumulation of tubulin subunits in the nucleus, leading to elevated CXCR4 transcription and increased chemotaxis of human HSCs. Treatment with microtubule stabilizer Epothilone B strongly suppresses the phenotypes induced by microtubule depolymerizing agents in human HSCs. Furthermore, chromatin immunoprecipitation assay reveals an increased binding of nuclear tubulin and TCF12 transcription factor at the CXCR4 promoter region. Depletion of TCF12 significantly suppresses microtubule depolymerization mediated upregulation of CXCR4 surface expression. These results demonstrate a previously unknown function of nuclear tubulin in regulating gene transcription through TCF12. New strategy targeting nuclear tubulin-TCF12-CXCR4 axis may be applicable to enhance HSC transplantation.

A genetic disorder reveals a hematopoietic stem cell regulatory network co-opted in leukemia

The molecular regulation of human hematopoietic stem cell (HSC) maintenance is therapeutically important, but limitations in experimental systems and interspecies variation have constrained our knowledge of this process. Here, we have studied a rare genetic disorder due to MECOM haploinsufficiency, characterized by an early-onset absence of HSCs in vivo. By generating a faithful model of this disorder in primary human HSCs and coupling functional studies with integrative single-cell genomic analyses, we uncover a key transcriptional network involving hundreds of genes that is required for HSC maintenance. Through our analyses, we nominate cooperating transcriptional regulators and identify how MECOM prevents the CTCF-dependent genome reorganization that occurs as HSCs differentiate. We show that this transcriptional network is co-opted in high-risk leukemias, thereby enabling these cancers to acquire stem cell properties. Collectively, we illuminate a regulatory network necessary for HSC self-renewal through the study of a rare experiment of nature.

Induction of Human T Cell Development In Vitro with OP9-DL4-7FS Cells Expressing Human Cytokines.

For nearly a generation now, OP9-DL1 and OP9-DL4 cells have provided an efficient and reliable cell system to generate T cells from mouse and human hematopoietic stem cells (HSCs) and pluripotent stem cells. OP9-DL1 and OP9-DL4 were originally derived from the OP9 mouse bone marrow stromal cell line, which was transduced to ectopically express Delta-like 1 or 4 proteins, respectively. OP9-DL cells mimic the thymic microenvironment in that when cocultured with mouse or human (h) HSCs, they interact with and activate Notch receptors present on HSCs, required for T cell differentiation. The HSC/OP9-DL cocultures require additional cytokines that are necessary for survival and proliferation of hematopoietic cells. For hHSCs, these factors are interleukin-7 (IL-7), stem cell factor (SCF), and FMS-like tyrosine kinase 3 ligand (FLT3L) that are normally exogenously added to the cocultures. In this chapter, we describe methods for establishing a novel and improved version of OP9-DL4 cells, called OP9-DL4-7FS cells that circumvent the addition of these costly cytokines, by transducing OP9-DL4 cell line to express human IL-7, FLT3L, and SCF (7FS). Herein, we describe the protocol for the generation of OP9-DL4-7FS cells and the conditions for OP9-DL4-7FS/hHSC coculture to support T cell lineage initiation and expansion while comparing it to the now "classic" OP9-DL4 coculture. The use of OP9-DL4-7FS cell system will provide an improved and cost-effective method to the commonly used OP9-DL/HSC coculture for studying both mouse and human T cell development.

Modeling Premalignant Transformation of Hematopoietic Stem Cells in a Nanobioreactor in Microgravity

Microgravity coupled with increased radiation exposure aboard the ISS provides a unique environment to simulate and study response to injury, inflammatory signaling, aging, and (pre-)malignant transformation of normal human hematopoietic stem cells (HSCs) in an accelerated timeframe. The NASA Twin study suggests that genomic, epigenomic, epitranscriptomic, and proteomic changes may detrimentally impact hematopoietic stem and immune cell fitness and induce stem cell exhaustion (Garrett-Bakelman et al., Science, 2019). Moreover, changes indicative of pre-cancer stem cell generation, such as increased chromosome translocations and inversions, occurred and persisted post-flight. Additionally, a recent publication entitled Multisystem Toxicity in Cancer: Lessons from NASA's Countermeasures Program found significant similarities between the multisystem physiological toxicities experienced in cancer patients as well as during spaceflight (Scott et al, Cell 2019). These reports highlight the benefits of studying injury response, changes in mutational profiles and cancer evolution in microgravity at the stem cell level. For this study, we designed a novel bioreactor system to support the culture of donor-derived human HSCs in low Earth orbit (LEO). A sponge matrix and stromal cells model the microenvironment HSCs reside in within the bone marrow niche. Testing on Earth confirmed our system's ability to maintain stem cell fitness over several weeks. To assess stem cell physiology in LEO over time, we lentivirally transduced a reporter into the HSCs pre-flight (Pineda et al., Scientific Reports 2016), which allows for cell cycle tracking via fluorescence imaging. This will provide data for assessment of stem cell health, maintenance and functionality. Furthermore, we are analyzing cytokine profiles and mutational status post-flight, with a focus on signatures we have previously connected to (pre-)malignant transformation via RNA sequencing analysis (Jiang, Cancer Cell 2019). The first batch of bioreactors launched with the SpX-24 mission in December 2021 and the second batch is currently on orbit as part of the SpX-25 mission. This investigation has the potential to provide valuable insights into the maintenance of hematopoietic stem cell health and functionality, HSC response to injury through accumulation of mutations and, eventually, the mechanisms fueling long-term (pre-)malignant transformation into leukemia stem cells.

Abstract 3154: Assessing hematopoietic stem cell fitness within a nanobioreactor in microgravity

Abstract Microgravity coupled with increased radiation exposure aboard the ISS provides a unique environment to simulate and study response to injury, inflammatory signaling, aging, and (pre-)malignant transformation of normal human hematopoietic stem cells (HSCs) in an accelerated timeframe. The NASA Twins study suggests that genomic, epigenomic, epitranscriptomic, and proteomic changes may detrimentally impact hematopoietic stem and immune cell fitness and induce stem cell exhaustion (Garrett-Bakelman et al., Science, 2019). Moreover, changes indicative of pre-cancer stem cell generation such as increased chromosome translocations and inversions occurred and persisted post-flight. Additionally, a recent publication entitled Multisystem Toxicity in Cancer: Lessons from NASA’s Countermeasures Program found significant similarities between the multisystem physiological toxicities in cancer patients and during spaceflight (Scott et al, Cell 2019). These reports highlight the benefits of studying injury response, changes in mutational profiles and cancer evolution in microgravity at the stem cell level. For this study, we designed a novel bioreactor system to support the culture of donor-derived human HSCs in low Earth orbit (LEO). A sponge matrix and stromal cells model the microenvironment HSCs reside in within the bone marrow niche. Testing on Earth confirmed our system’s ability to maintain stem cell fitness over several weeks. To assess stem cell physiology in LEO over time, we lentivirally transduce a reporter into the HSCs pre-flight (Pineda et al., Scientific Reports 2016), which allows for cell cycle tracking via fluorescence imaging. This will provide data for assessment of stem cell health, maintenance and functionality. Furthermore, we will be analyzing mutational status post-flight, with a focus on signatures we have previously connected to (pre-)malignant transformation via RNA sequencing analysis (Jiang, Cancer Cell 2019). Our bioreactors are scheduled to launch as part of the SpaceX CRS-24 mission on Dec 21, 2021. This investigation may provide valuable insights into the maintenance of hematopoietic stem cell health and functionality, response to injury through accumulation of mutations and, eventually, the mechanisms fueling long-term (pre-)malignant transformation into leukemia stem cells. Citation Format: Luisa Ladel, Jessica Pham, Isabelle Oliver, Larisa Balaian, Catriona HM Jamieson. Assessing hematopoietic stem cell fitness within a nanobioreactor in microgravity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3154.

Challenges in Cell Fate Acquisition to Scid-Repopulating Activity from Hemogenic Endothelium of hiPSCs Derived from AML Patients Using Forced Transcription Factor Expression.

The generation of human hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) represents a major goal in regenerative medicine and is believed would follow principles of early development. HSCs arise from a type of endothelial cell called a “hemogenic endothelium” (HE), and human HSCs are experimentally detected by transplantation into SCID or other immune-deficient mouse recipients, termed SCID-Repopulating Cells (SRC). Recently, SRCs were detected by forced expression of seven transcription factors (TF) (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, and SPI1) in hPSC-derived HE, suggesting these factors are deficient in hPSC differentiation to HEs required to generate HSCs. Here we derived PECAM-1-, Flk-1-, and VE-cadherin-positive endothelial cells that also lack CD45 expression (PFVCD45−) which are solely responsible for hematopoietic output from iPSC lines reprogrammed from AML patients. Using HEs derived from AML patient iPSCs devoid of somatic leukemic aberrations, we sought to generate putative SRCs by the forced expression of 7TFs to model autologous HSC transplantation. The expression of 7TFs in hPSC-derived HE cells from an enhanced hematopoietic progenitor capacity was present in vitro, but failed to acquire SRC activity in vivo. Our findings emphasize the benefits of forced TF expression, along with the continued challenges in developing HSCs for autologous-based therapies from hPSC sources.

Le statu quo n’est plus une option

<h3>ABSTRACT</h3> Sickle Cell Disease (SCD), one of the world’s most common genetic disorders, causes anemia and progressive multiorgan damage that typically shortens lifespan by decades; currently there is no broadly applicable curative therapy. Here we show that Cas9 RNP-mediated gene editing with an ssDNA oligonucleotide donor yields markerless correction of the sickle mutation in more than 30% of long-term engrafting human hematopoietic stem cells (HSCs), using a selection-free protocol that is directly applicable to a clinical setting. We further find that <i>in vivo</i> erythroid differentiation markedly enriches for corrected ß-globin alleles. Adoption of a high-fidelity Cas9 variant demonstrates that this approach can yield efficient editing with almost no off-target events. These findings indicate that the sickle mutation can be corrected in human HSCs at curative levels with a streamlined protocol that is ready to be translated into a therapy. <h3>ONE SENTENCE SUMMARY</h3> Cas9-mediated correction of the sickle mutation in human hematopoietic stem cells can be accomplished at curative levels.

ADGRG1 enriches for functional human hematopoietic stem cells following ex vivo expansion-induced mitochondrial oxidative stress.

The heterogeneity of human hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) under stress conditions such as ex vivo expansion is poorly understood. Here, we report that the frequencies of SCID-repopulating cells were greatly decreased in cord blood (CB) CD34+ HSCs and HPCs upon ex vivo culturing. Transcriptomic analysis and metabolic profiling demonstrated that mitochondrial oxidative stress of human CB HSCs and HPCs notably increased, along with loss of stemness. Limiting dilution analysis revealed that functional human HSCs were enriched in cell populations with low levels of mitochondrial ROS (mitoROS) during ex vivo culturing. Using single-cell RNA-Seq analysis of the mitoROS low cell population, we demonstrated that functional HSCs were substantially enriched in the adhesion GPCR G1-positive (ADGRG1+) population of CD34+CD133+ CB cells upon ex vivo expansion stress. Gene set enrichment analysis revealed that HSC signature genes including MSI2 and MLLT3 were enriched in CD34+CD133+ADGRG1+ CB HSCs. Our study reveals that ADGRG1 enriches for functional human HSCs under oxidative stress during ex vivo culturing, which can be a reliable target for drug screening of agonists of HSC expansion.

An Enhanced Zebrafish Xenograft Model to Study the Behavior of Human Hematopoietic Stem Cells and Patient Derived Leukemia In Vivo

Human hematopoietic stem cells (HSCs) are characterized by their unique ability to self-renew and differentiate into multiple cell types. Transplantation of human HSCs into immunocompromised mice is the gold standard for evaluating the in vivo repopulation capacity of these cells and has been adapted to evaluate the leukemic potential of leukemia-initiating cells (LICs) in acute leukemia. In spite of the popularity of this approach, certain cellular subsets do not survive post xenotransplantation as they are dependent on human factors that may be missing in the murine microenvironment. We pioneered human leukemia xenografts in the zebrafish model (Corkery et al., 2011; Bentley et al., 2015) as a parallel strategy, taking advantage of the immuno-permissiveness and transparency of zebrafish larvae to provide unprecedented continuous observation of leukemia evolution. However, as in mice, the addition of human cytokines and growth factors may further improve engraftment and optimize the utility of the zebrafish as a preclinical transplant model for medium to high-throughput therapeutic screening by creating a microenvironment more comparable to that found in patients.Using transgenic technology, we created novel zebrafish lines that express human stem cell factor (SCF/KITLG), granulocyte-macrophage colony stimulating factor (GM-CSF/CSF2), and stromal cell-derived factor 1 alpha (SDF1α/CXCL12). All of these factors are essential for the survival and expansion of HSCs, progenitors and LICs. CMK, GRWi (Down Syndrome- acute myeloid leukemia (DS-AML)) and Jurkat (T-acute lymphoblastic leukemia) cell lines were injected into the yolk sac of these multi-cytokine zebrafish embryos. These cells engrafted and exhibited increased cell proliferation and physiological migration compared to control casper embryo transplants. Moreover, only in the multi-cytokine fish did these cells home to hematopoietic niches, including the caudal hematopoietic tissue (CHT) (equivalent to the fetal liver) at 7 days-post injection (dpi) and the kidney marrow at 11 dpi.The yolk sac is not the physiological site for hematopoietic cell growth and thus we injected patient-derived AML cells into the circulation of the multi-cytokine fish. We observed similar trends in proliferation and migration as seen with leukemia cell lines. Historically, human HSCs do not survive beyond 24h in zebrafish xenografts (Pruvot et al. 2011). Given our success with patient-derived leukemia samples, we wanted to see if our multi-cytokine fish would provide a more favorable environment for human HSC survival. Indeed, primary human umbilical cord blood-derived HSCs were injected into the circulation of zebrafish larvae and survived past 3 dpi in the multi-cytokine fish but not in casper control larvae.Transgenic zebrafish expressing human cytokines provide an enhanced model for studying human leukemia and stem cell biology in a visually tractable system that more closely recapitulates the human microenvironment. The inherent capacity of the zebrafish for rapid and efficient chemical screening will be exploited in this model for preclinical testing of anti-leukemic therapies and to evaluate HSC enhancing agents in vivo . DisclosuresBerman:AGADA Therapeutics: Research Funding.

Molecular Insights at the Single Cell Level into the Reprogramming of Human Hematopoietic Stem Cells during Epigenetically Modified Ex Vivo Expansion

The rarity of human hematopoietic stem cells (HSCs) and the very limited ability to study these cells in vivo in the human stem cell niche are significant hurdles in understanding the regulatory networks and cues that control the balance between human HSC maintenance, survival, quiescence, self-renewal and differentiation. In murine studies, epigenetic mechanisms have been demonstrated to play important roles in regulating HSC fate (Cullen SM et al. Curr Top Dev Biol. 2014; Wang et. al. Cell Stem Cell. 2016). One key mechanism involves histone modification by acetylation, with the acetylation of histones and non-histone proteins being controlled by histone acetyltransferases and histone deacetylates (HDAC). Recent studies have demonstrated that combining histone deacetylase inhibitors (HDACi), with specific hematopoietic cytokine priming under serum-free conditions can significantly enhance the expansion ex vivo of phenotypically defined and/or NSG mouse engraftable human HSC (SCID repopulating ability cells or SRC) from cord blood (CB). Indeed, ex vivo expansion with VPA in combination with FL, TPO, SCF and IL-3 for 7 days was reported to enhance human cord blood SRCs by almost 300 fold more than cytokine priming alone and 35.9 fold more than unexpanded cord blood (Chaurasia et. al. J Clin Invest. 2014). Subsequent microarray analyses of bulk CD34+ hematopoietic progenitor cells (HSPCs) after VPA plus cytokine treatment were used to define epigenetic signatures (Gajzer et. al. Vox Sang. 2016).Given that human phenotypically-defined HSCs (Lin-CD34+CD38-CD45RA-CD90+CD49f+) or long-term (LT) SRCs would be expected to increase 3-5 fold over 7 day cultures (estimated median doubling time of 36-48 hours), that LT-SRC are characterised by delayed G0 exit (with 1st division averaging 66-75h), that short-term SRC proliferate more rapidly, that HSCs are contained in both the CD90+ and CD90- subsets and that HSC develop in micro-environmental niches which provide additional regulatory cues (Laurenti E et al. Cell Stem Cell. 2015; Knapp et. al. Stem Cell Reports. 2017; Zonari E et al. Stem Cell Reports. 2017), we hypothesised that HDACi in the presence of cytokines might not only expand the CD90+ subset without differentiation but also reprogram CD90- cells to CD90+ HSCs.To test this hypothesis, we first cultured human CB CD133+ cells in SCF, FL and TPO with HDACi or vehicle addition on Nanex scaffolds (Gullo et. al. Bioinformatics. 2015). Total cell expansion over 5 days was consistently lower in HDACi cultures compared to the vehicle control, with HDACi significantly increasing the absolute numbers of phenotypically-defined HSCs. The HDACi-induced increase of HSCs was accompanied by a concomitant decrease of phenotypically defined Lin-CD38-CD34+CD45RA-CD90- cells. To confirm that cells within the CD90- population were able to give rise to CD90+ phenotypically-defined LT-HSC, we then sorted Lin-CD34+CD38-CD45RA-CD90- cells into serum-free medium containing cytokines and HDACi. After 2-day culture, the majority of CD90- cells became CD90+ culture (with no significant up-regulation of CD49f). Next, we sorted CD133+ cells, cultured for 5 days in cytokines with vehicle or HDACi, into CD90+ or CD90- subsets (Lin-CD34+CD38-CD45RA-CD49f+) and analysed their transcriptomes (100 cells per sample from 2 different donors) by RNA-sequencing using SMART-seq2 (Picelli et. al. Nature Methods. 2014). RNA-sequencing data of CD90+ cells expanded with vehicle or HDACi did not show statistically significant differences in their gene expression. However, we identified 55 genes that were differently expressed between CD90+ and CD90- (Lin-CD34+CD38-CD45RA-CD49f+) subsets but unchanged in the vehicle and HDACi conditions. Using single cell sorting and Fluidigm Biomark technology, we confirmed the differential expression of these genes between CD90+ and CD90- cells (Lin-CD34+CD38-CD45RA-CD49f+). In conclusion, we have defined gene signatures for human CD90+ and CD90- (Lin-CD34+CD38-CD45RA-CD49f+) subsets after treatment with the HDACi, and demonstrate that HDACi does not alter gene expression of the CD90+ subset but is able to both delay HSC differentiation and reprogram CD90- cells into CD90+ cells during in vitro culture. DisclosuresMead:BMS: Honoraria; Pfizer: Honoraria; Novartis: Honoraria, Research Funding, Speakers Bureau.

Engineering Precision Medicine to Enhance Graft-Versus-Lymphoma Activity: Hematopoietic Stem Cells Modified with Chimeric Antigen Receptors

Background: Gene-modified human hematopoietic stem cells (HSC) have been used to introduce genes to correct, prevent or treat diseases. Clinical trials using infusion of autologous T cells engineered with anti-CD19 chimeric antigen receptors (CAR) have demonstrated complete remissions even in chemotherapy-resistant malignancies, but the persistence of the effector cells is transient, limiting clinical efficacy and allowing the cancer to recur. Current trials of CAR T cells have recommended patients in remission post-T cell therapy to undergo hematopoietic stem cell transplantation (HSCT) due to the limited persistence of the CAR+ T cells. Seeking to foster persistence of the CAR-modified immune cells, we propose using gene modification of HSC to create persistent generation of multilineage immune effectors to directly target cancer cells. Concerns regarding malignant transformation, abnormal hematopoiesis and autoimmunity exist, making the co-delivery of a suicide gene a necessary safety measure. We evaluated a second-generation anti-CD19 chimeric antigen receptor (CAR) to human HSC in a humanized mouse model, co-delivered with a truncated epidermal growth factor receptor (EGFRt) as a suicide gene system.Significance: CAR+ HSC can be infused in the context of HSCT, when the chemotherapy-conditioning regimen favors engraftment of gene-modified cells, decreases residual tumor burden and decreases the chance of CAR immunogenicity. The prospect of modifying autologous cells to enhance GVL bears the possibility of decreased morbidity and mortality, a concern for specifically vulnerable populations such as children and elderly patients. In addition, this approach will offer alternative therapy for those without cell sources available for allogeneic HSCT, benefiting ethnic minorities.Methods: Third-generation self-inactivating lentiviral vectors were used to co-deliver an anti-CD19 CAR and EGFRt. In vitro, gene-modified HSC were differentiated into myeloid cells to allow transgene expression. Antibody-dependent cell-mediated cytotoxicity (ADCC) assay was used incubating target cells with leukocytes and monoclonal antibody cetuximab to determine percentage of surviving cells. In vivo, gene-modified HSC were engrafted into immunodeficient NSG mice treated or not with intraperitoneal cetuximab 1mg/mouse/day for 12 days. Persistence of gene-modified cells was assessed by flow cytometry, ddPCR, and PET imaging using 89Zr-Cetuximab.Results: Gene modification of HSC did not affect engraftment, proliferation or differentiation, at efficiency of 20-40%. Antigen specificity of myeloid, NK and T cells were successfully redirected against CD19. Ablation of gene-modified cells was significantly increased (p=0.01) in target cells expressing EGFRt after incubation with leukocytes and cetuximab 1µg/mL, compared to EGFRt+ cells without cetuximab, and non-transduced cells with or without cetuximab. This was seen at all effector-to-target ratios. Mice engrafted with CAR-modified HSC presented prolonged persistence of gene-modified cells in bone marrow, spleen, thymus and peripheral blood, and CAR+ cells elicited protection against CD19+ tumors, with inhibition or elimination of tumor development and survival advantage. Mice humanized with gene-modified HSC for the expression of the suicide gene had significant ablation of gene-modified cells after treatment with cetuximab (p=0.002). Remaining gene-modified cells were close to background on flow cytometry and within two logs of decrease of vector copy numbers by ddPCR in mouse tissues. PET imaging of humanized mice documented engraftment of EGFRt-expressing cells in bone marrow, spleen and liver, and confirmed ablation with an average decrease of 82.5% after cetuximab treatment.Conclusions: These results give proof of principle for persistence and anti-tumor activity of CAR-modified HSC regulated by suicide gene, and further studies are needed to enable clinical translation. Cetuximab ADCC of EGFRt-modified cells caused effective killing. Different ablation approaches, such as inducible caspase 9 or co-delivery of other inert cell markers should be evaluated. DisclosuresNo relevant conflicts of interest to declare.

3D Multicellular Spheroid for the Study of Human Hematopoietic Stem Cells: Synergistic Effect Between Oxygen Levels, Mesenchymal Stromal Cells and Endothelial Cells.

IntroductionThe human bone marrow microenvironment is composed of biological, chemical and physical factors that act in a synergistic way to modulate hematopoietic stem cell biology, such as mesenchymal stromal cells (MSCs), endothelial cells (ECs) and low oxygen levels; however, it is difficult to mimic this human microenvironment in vitro.MethodsIn this work, we developed 3D multicellular spheroid (3D-MS) for the study of human hematopoietic stem cells (HSCs) with some components of perivascular niche. HSCs were isolated from umbilical cord blood, MSCs were isolated from human bone marrow and a microvasculature EC line (CC-2811, Lonza®) was used. For the formation of a 3D structure, a magnetic levitation culture system was used. Cultures were maintained in 21%, 3% and 1% O2 for 15 days. Culture volume, sphericity index and cell viability were determined. Also, human HSC proliferation, phenotype and production of reactive oxygen species were evaluated.ResultsAfter 15 days, 3D-MS exhibited viability greater than 80%. Histology results showed structures without necrotic centers, and higher cellular proliferation with 3% O2. An increase in the expression of the CD34 antigen and other hematopoietic antigens were observed to 1% O2 with MSCs plus ECs and low ROS levels.ConclusionThese findings suggest that 3D-MS formed by MSCs, ECs and HSCs exposed to low concentrations of oxygen (1–3% O2) modulate human HSC behavior and mimics some features of the perivascular niche, which could reduce the use of animal models and deepen the relationship between the microenvironment of HSC and human hematological diseases development.

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells.

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.