In vitro differentiation of human pluripotent stem cells into the B lineage using OP9-MS5 co-culture.

In vitro differentiation of human pluripotent stem cells (hPSCs) offers a genetically tractable system to examine the physiology and pathology of human tissue development and differentiation. We have used this approach to model the earliest stages of human B lineage development and characterize potential target cells for the in utero initiation of childhood B acute lymphoblastic leukemia. Herein, we detail critical aspects of the protocol including reagent validation, controls, and examples of surface markers used for analysis and cell sorting. For complete details on the use and execution of this protocol, please refer to Boiers et al. (2018).Wellcome Trust, Cancer Research UK, Swedish Research Council, Swedish Childhood Cancer Foundatio

TLR7 ligation augments hematopoiesis in Rps14 (uS11) deficiency via paradoxical suppression of inflammatory signaling

Myelodysplastic syndrome (MDS) is a hematological malignancy characterized by blood cytopenias and predisposition to acute myeloid leukemia (AML). Therapies for MDS are lacking, particularly those that have an impact in the early stages of disease. We developed a model of MDS in zebrafish with knockout of Rps14, the primary mediator of the anemia associated with del(5q) MDS. These mutant animals display dose- and age-dependent abnormalities in hematopoiesis, culminating in bone marrow failure with dysplastic features. We used Rps14 knockdown to undertake an in vivo small-molecule screening, to identify compounds that ameliorate the MDS phenotype, and we identified imiquimod, an agonist of Toll-like receptor-7 (TLR7) and TLR8. Imiquimod alleviates anemia by promoting hematopoietic stem and progenitor cell expansion and erythroid differentiation, the mechanism of which is dependent on TLR7 ligation and Myd88. TLR7 activation in this setting paradoxically promoted an anti-inflammatory gene signature, indicating cross talk via TLR7 between proinflammatory pathways endogenous to Rps14 loss and the NF-κB pathway. Finally, in highly purified human bone marrow samples from anemic patients, imiquimod led to an increase in erythroid output from myeloerythroid progenitors and common myeloid progenitors. Our findings have both specific implications for the development of targeted therapeutics for del(5q) MDS and wider significance identifying a potential role for TLR7 ligation in modifying anemia

Initial seeding of the embryonic thymus by immune-restricted lympho-myeloid progenitors

The final stages of restriction to the T cell lineage occur in the thymus after the entry of thymus-seeding progenitors (TSPs). The identity and lineage potential of TSPs remains unclear. Because the first embryonic TSPs enter a non-vascularized thymic rudiment, we were able to directly image and establish the functional and molecular properties of embryonic thymopoiesis-initiating progenitors (T-IPs) before their entry into the thymus and activation of Notch signaling. T-IPs did not include multipotent stem cells or molecular evidence of T cell-restricted progenitors. Instead, single-cell molecular and functional analysis demonstrated that most fetal T-IPs expressed genes of and had the potential to develop into lymphoid as well as myeloid components of the immune system. Moreover, studies of embryos deficient in the transcriptional regulator RBPJ demonstrated that canonical Notch signaling was not involved in pre-thymic restriction to the T cell lineage or the migration of T-IPs

A Human IPS Model Implicates Embryonic B-Myeloid Fate Restriction as Developmental Susceptibility to B Acute Lymphoblastic Leukemia-Associated ETV6-RUNX1

ETV6-RUNX1 is associated with childhood acute B-lymphoblastic leukemia (cALL) functioning as a first-hit mutation that initiates a clinically silent pre-leukemia in utero. Because lineage commitment hierarchies differ between embryo and adult, and the impact of oncogenes is cell-context dependent, we hypothesized that the childhood affiliation of ETV6-RUNX1 cALL reflects its origins in a progenitor unique to embryonic life. We characterize the first emerging B cells in first-trimester human embryos, identifying a developmentally restricted CD19-IL-7R+ progenitor compartment, which transitions from a myeloid to lymphoid program during ontogeny. This developmental series is recapitulated in differentiating human pluripotent stem cells (hPSCs), thereby providing a model for the initiation of cALL. Genome-engineered hPSCs expressing ETV6-RUNX1 from the endogenous ETV6 locus show expansion of the CD19-IL-7R+ compartment, show a partial block in B lineage commitment, and produce proB cells with aberrant myeloid gene expression signatures and potential: features (collectively) consistent with a pre-leukemic state

The Road to Maturity - Lineage Commitment in early Hematopoiesis

The road to maturity – how do hematopoietic stem cells (HSC) differentiate into mature blood cells? The pathways of lineage commitment during normal hematopoiesis are of great significance in order to understand the underlying events that lead to leukemia, and to the design of proper treatments for prevention and remission of the disease. The route of hematopoiesis can be thought of as a hierarchical tree, with the rare HSCs at the top, transitioning down along the pathways as different progenitors. These progenitors continue on their way to become mature blood cells. The roads of blood cell production have been extensively studied in the adult mouse model, whereas less is known about the differentiation of cells during fetal mapping. It is not even known if blood cell commitment follows the same route in adult as in fetal life. The fetal map may be even more important to study since some pre-leukemic events are of prenatal origin. This thesis focuses on the role of different cytokines in lineage commitment, as well as on identifying the first lymphoid committed progenitor in the early fetus. The c-fms like tyrosine kinase 3 receptor (Flt3) is known to be important for lymphopoiesis. However, Flt3 is often found mutated in acute myeloid leukemia (AML), and then associated with poor prognosis. Despite the role in AML, no role for Flt3 or its ligand has been found in myelopoiesis. But more distinct stages of early myeloid progenitors can now be identified, and the role of Flt3 in myelopoiesis could be investigated in detail. We found that early myeloid progenitors express high levels of Flt3, and in mice deficient in Flt3 signaling myeloid progenitors are reduced. Taken together the data clearly show a role of Flt3 signaling in early myelopoiesis, which has implications for understanding the role of Flt3 mutations in AML. In the next study the key cytokines in B lymphopoiesis were investigated. The role of Flt3 signaling together with interleukin 7 (IL7) and a cytokine called Thymic stromal lymphopoietin (TSLP) was studied. TSLP has been suggested to have a key role in IL7 independent B cell development, although direct evidence has been lacking. By using different knockout mice the role of the three signaling pathways was investigated side by side in fetal and adult mice. Mice deficient in all three signaling pathways lacked B cells, as did mice deficient in IL7 and Flt3 signaling. The conclusion is that the IL7 and Flt3 signaling pathways are the main factors driving both fetal and adult B lymphopoiesis. In fetal hematopoiesis, as mentioned previously, the road to maturity is not well understood. We aimed at identifying the first lymphoid commitment step in the early embryo. A population in the fetal liver at 11.5 days post coitus (dpc), expressing the markers Flt3 and IL7 receptor alfa (IL7Rα), was purified. It was shown to have combined lymphoid and granulocyte/macrophage potential but no megakaryocyte or erythroid potential at the single cell level. This population could represent the first lympho-myeloid restricted cells in ontogeny, and further evidence suggests that it might be the first progenitor that seeds the thymus. By using a reporter mouse for recombination activating gene 1, Rag1, (an early sign of lymphoid commitment), a lympho-myeloid restricted population, expressing IL7Rα and Rag1-GFP, could be traced back to 9.5 dpc. Fetal liver colonization has been shown to begin at 9.5 dpc and definitive adult HSCs appear first one day later at 10.5 dpc. This study identifies a lymphomyeloid restricted progenitor in the early embryo, notably arising prior to the establishment of definitive HSCs, and suggests that lymphoid commitment might take place outside the fetal liver niche

Defining the Emerging Blood System During Development at Single-Cell Resolution

Developmental hematopoiesis differs from adult and is far less described. In the developing embryo, waves of lineage-restricted blood precede the ultimate emergence of definitive hematopoietic stem cells (dHSCs) capable of maintaining hematopoiesis throughout life. During the last two decades, the advent of single-cell genomics has provided tools to circumvent previously impeding characteristics of embryonic hematopoiesis, such as cell heterogeneity and rare cell states, allowing for definition of lineage trajectories, cellular hierarchies, and cell-type specification. The field has rapidly advanced from microfluidic platforms and targeted gene expression analysis, to high throughput unbiased single-cell transcriptomic profiling, single-cell chromatin analysis, and cell tracing-offering a plethora of tools to resolve important questions within hematopoietic development. Here, we describe how these technologies have been implemented to address a wide range of aspects of embryonic hematopoiesis ranging from the gene regulatory network of dHSC formation via endothelial to hematopoietic transition (EHT) and how EHT can be recapitulated in vitro, to hematopoietic trajectories and cell fate decisions. Together, these studies have important relevance for regenerative medicine and for our understanding of genetic blood disorders and childhood leukemias

In vitro differentiation of human pluripotent stem cells into the B lineage using OP9-MS5 co-culture

Summary: In vitro differentiation of human pluripotent stem cells (hPSCs) offers a genetically tractable system to examine the physiology and pathology of human tissue development and differentiation. We have used this approach to model the earliest stages of human B lineage development and characterize potential target cells for the in utero initiation of childhood B acute lymphoblastic leukemia. Herein, we detail critical aspects of the protocol including reagent validation, controls, and examples of surface markers used for analysis and cell sorting.For complete details on the use and execution of this protocol, please refer to Boiers et al. (2018)

Effect of high-fat diet, surrounding temperature, and enterostatin on uncoupling protein gene expression

Nonshivering thermogenesis induced in brown adipose tissue (BAT) during high-fat feeding is mediated through uncoupling protein 1 (UCP1). UCP2 is a recently identified homologue found in many tissues. To determine the role of UCP1 and UCP2 in thermoregulation and energy balance, we investigated the long-term effect of high-fat feeding on mRNA levels in mice at two different ambient temperatures. We also treated mice with the anorectic peptide enterostatin and compared mRNA levels in BAT, white adipose tissue (WAT), stomach, and duodenum. Here, we report that high-fat feeding at 23 degrees C increased UCP1 and UCP2 levels in BAT four- and threefold, respectively, and increased UCP2 levels fourfold in WAT. However, at 29 degrees C, UCP1 decreased, whereas UCP2 remained unchanged in BAT and increased twofold in WAT. Enterostatin increased UCP1 and decreased UCP2 mRNA in BAT. In stomach and duodenum, high-fat feeding decreased UCP2 mRNA, whereas enterostatin increased it. Our results suggest that the regulation of uncoupling protein mRNA levels by high-fat feeding is dependent on ambient temperature and that enterostatin is able to modulate it