HSC-like Cells Research Articles

Stem-Cell Enriched Cellular Hierarchy of TP53 Mutant Acute Myeloid Leukemia Is Vulnerable to Targeted Protein Degradation of c-MYC

Deregulation of MYC genes occurs in up to 70% of all human cancers and is associated with hallmarks of cancer including mitochondrial and ribosomal biogenesis, cell cycle progression, and metabolic abnormalities. TP53 regulates MYC while MYC suppresses TP53, suggesting counteracting negative feedback loops. Therefore, MYC or its function can be activated when TP53 is not functional. TP53 mutations occur in 30% of relapsed/refractory acute myeloid leukemias (AMLs) patients' survival is dismal, and there are no effective therapies for these patients. Compared to TP53 wild-type (TP53wt), TP53 mutant (TP53mut) AMLs have lower percentages and numbers of leukemia blasts with increased immature CD34+ cells and resistance to chemo- or molecularly targeted therapies. However, the exact cellular hierarchy of TP53mut AML has not been elucidated. We observed significantly increased MYC mRNA levels in (TP53mut), as compared to TP53wt AML, and also increased levels in TP53mut versus TP53wt AML leukemia stem cell (LSC) fractions. This finding was confirmed in a dataset from the Munich Leukemia Laboratory (N = 732). We found significantly upregulated MYC pathways in TP53mut compared to TP53wt AML LSC. We confirmed the increased MYC mRNA levels at the protein level in TP53mut AML by single-cell mass cytometry (CyTOF). To dissect the cellular hierarchy in TP53mut AML, we performed single-cell RNA sequencing of 32 BM samples from healthy donors (N = 3), newly diagnosed, high-risk TP53wt (N = 7) and TP53mut (N = 22) AML patients, with 6,685, 10,687 and 10,687 cells from normal, TP53wt and TP53mut AML bone marrow (BM) cells, respectively. We found highly enriched HSC-like cells and reduced progenitor- and GMP-like cells in TP53mut AML compared to normal BM (NBM) and TP53wt AML samples. We overlayed MYC expression levels on the mapping of cellular components and found higher MYC levels in HSC-like cells in TP53mut AML compared to HSC and HSC-like cells in NBM and TP53wt AML samples, suggesting enrichment of immature HSC-like cells and increased activity of MYC in TP53mut AML LSCs ( Fig. A). To target c-MYC and MYC signaling, we utilized GT19715, the first-in-class cereblon modulator (CELMoD) for c-MYC protein (Nishida, ASH 2022). CyTOF confirmed the presence of much increased c-MYC protein levels in primary CD34+ AML than in CD34+ NBM hematopoietic stem cells (HSCs). Notably, CD34+ AML cells showed much greater sensitivity to GT19715 compared to CD34+ NBM cells. Data suggest on-target activity of GT19715 against c-MYC in LSCs with a therapeutic window between LSCs vs. NBM HSCs. Intriguingly, c-MYC protein levels are higher in CD34+CD38+ than in CD34+CD38- AML cells, suggesting that c-MYC drives the proliferation of AML progenitor cells differentiating from quiescent LSCs. Consequently, CD34+CD38+ AML progenitor cells exhibited greater sensitivity to GT19715 compared to CD34+CD38- LSCs. Using paired, TP53 null HL-60 GT19715-sensitive and -resistant cells generated through chronic exposure to GT19715, we interrogated the impact of GT19715 on metabolic changes. GT19715 induced pronounced reductions in basal and maximal oxygen consumption rates (OCRs) in GT19715-sensitive HL-60 cells. GT19715 reduced both basal, glutamine- and glucose-dependent extracellular acidification rates (ECARs) in GT19715-sensitive cells while no inhibition in OCRs or ECARs was observed in GT19715-resistant cells. GT19715 severely inhibited ECARs even after adding glucose to GT19715-sensitive cells, suggesting irreversible inhibition of glycolysis as one of the mechanisms of action of GT19715. GT19715 profoundly reduced AML blasts in TP53mut AML samples (N = 3) ( Fig. B), and in a very aggressive patient-derived xenograft (PDX) TP53mut AML model established from a patient with TP53 p.Y220C and p.P151A mutations along with MECOM rearrangement and K/NRAS mutations. In humanized Crbn I391V mice, where the Crbn-mediated protein degradation is operational, GT19715 only reduced WBC counts along with minimal body weight loss. GT19715 but did not reduce total mouse BM CD45+ cells, suggesting favorable toxicity profiles of GT19715. In conclusion, TP53mut AML comprised highly enriched LSC populations compared to TP53wt AML and targeting of c-MYC protein is highly effective in TP53mut AML in vitro and in vivo with a therapeutic window between AML LSC and normal hematopoietic cells.

Multi-Omics Factor Analysis (MOFA) Identifies Transposable Element Expression As a Risk Factor and Inflammaging As a Protective Factor in Myelodysplastic Syndromes

Introduction Myelodysplastic syndromes (MDS) are haematological diseases characterised by clonal proliferation due to genetic and epigenetic alterations within haematopoietic stem and progenitor cells. Thus far, the prediction of a patient's overall survival (OS) and event-free survival (EFS) has been dependent predominantly on the degree of peripheral blood cytopenias, bone marrow blast percentage, cytogenetics, and genetic features. However, this may overlook other biological phenotypes, including, for example, the expression of transposable elements (TEs), ageing, and immunology profile. In this study, we present a comprehensive analysis of three data modalities (clinical, genotypic, and transcriptomic) and eight different views derived from these modalities to identify the hidden factors that significantly impact MDS prognosis. Methods Three (immunology, ageing, and cellular composition) out of eight views of the data were derived from RNA-seq by applying singscore, a gene set scoring tool developed by Foroutan et al., 2018, on RNA-seq gene expression from bone marrow samples of 94 MDS patients obtained from Shiozawa et al., 2017 (Fig 1.A). Each of these three views was carried forward in the workflow by a number of gene sets. The other five views were clinical numeric data, clinical categorical data, TE expression, genotype data, and known AML/MDS antigens (obtained from TANTIGEN 2.0), expressed in at least 10% of MDS samples in the study. We then employed Multi-Omics Factor Analysis (MOFA), a computational framework developed by Argelaguet et al., 2018, to uncover hidden phenotypic states within a multi-omic dataset. Through MOFA, we generated factors representing specific biological data modalities, establishing connections between features across these different categories. The total variance ( R2) that can be attributed to specific factors is also exhibited by MOFA. Results Factors were ranked based on their explained variance. We present the 10 factors with a threshold of variance explained of 2% from the 15 default factors generated by MOFA (Fig 1.A). Factor 1 was one of the factors that linked the most categories of data: immunology, cellular composition, ageing, and TEs. Most notably, Factor 1 was aligned with features of less differentiated cells (more HSC-like and less GMP-like). We observed that samples represented by high Factor 1 scores also had a higher expression of SINE:Alu elements. (Fig 1.A). Moreover, Factor 1 aligns the following immunology features: increased T-helper 1 (Th1) cells, macrophages, and T regulatory cells. The relationship between Th1 and SINE elements is particularly interesting, given the role of Th1 cell activation in host defence against infection that may be modulated by SINE activation. Factor 2 comprised of ageing and clinical categorical data, which showed that less inflammaging may lead to a poorer EFS ( P=0.00023) (Fig 1.B). Factor 4 linked immunology, cellular decomposition, ageing and clinical categorical data. The following features mainly influenced Factor 4: increased HSC-like cells, decreased CD8+ T cells, decreased immunosenescence and increased inflammatory chemokines. Using Kaplan-Meier plots to predict OS, we observed that patients with more HSC-like cells, fewer leukocytes and T cells, and less immunosenescence and inflammaging were linked to poorer EFS ( P=0.0001) and OS ( P=0.00017, data not shown). Furthermore, Factor 9 comprised solely of the TEs category and exhibited a correlation between increased TE expression and poorer EFS ( P=0.0074) and OS ( P=0.0065, data not shown) (Fig 1.B). Conclusions Our analysis using MOFA on a diverse set of biological data modalities uncovered hidden factors in MDS, providing insights into the complex interplay between various biological layers. Notably, we identified TE expression as a risk factor and inflammaging as a protective factor in MDS. HSC-like cells and specific immunological profiles were also associated with significantly poor OS and EFS. This study contributes to a deeper understanding of MDS pathogenesis and identifies potential prognostic markers for this disease. It also elucidates the importance of considering the relationships between different pathways, markers, and mutations in predicting patient outcomes, highlighting the efficacy of a comprehensive approach that goes beyond all the scoring systems described thus far for MDS.

Differentiation State Plasticity As a Mechanism of BCL2 Inhibitor Resistance in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is composed of a heterogeneous blast population that exhibits characteristics of normal myeloid cells, including immature, HSC-like cells and differentiated, monocyte-like cells. The balance of immature and differentiated blasts within a patient is important clinically, as immature AML blasts are acutely sensitive to treatment with venetoclax, a BH3 mimetic that blocks the anti-apoptotic activity of BCL2. BCL2-inhibitor-based therapeutic strategies are the standard of care among elderly patients with untreated or relapsed/refractory AML. While these strategies result in a remission rate of nearly 70%, the majority of patients who do achieve remission ultimately relapse. Frequently, the relapsed disease is composed of differentiated, monocyte-like blasts. Given the importance of BCL2 inhibitors to the clinical management of AML, it is critical to understand the mechanisms by which BCL2 inhibitors result in monocytic relapse. Disease relapse has been traditionally thought to emerge from intrinsically resistant cells that confer a survival advantage under therapeutic pressure. Several studies have suggested that differentiated AML blasts have a survival advantage in the presence of BCL2 inhibition due to a reliance on anti-apoptotic proteins other than BCL2. Immature AML blasts express high levels of BCL2, whereas blasts that are primed for lympho-myeloid or granulocytic-monocytic progenitor cell fates express high levels of MCL-1 and BCL-XL, respectively. However, these studies do not explain why patients with dominant, differentiated subclones frequently achieve long-lasting remission with BCL2-inhibitor-based strategies. Therefore, simple selection of differentiated blasts appears to be an insufficient model to describe BCL2 inhibitor resistance. Alternative models of therapeutic resistance have been identified where cancer cells adapt to a resistant phenotype and acquire a survival advantage under therapeutic pressure. In the context of BCL2 inhibitor resistance, it is unclear the extent to which the emergence of differentiated blasts is the result of selective pressure or blast differentiation state plasticity in response to BCL2 inhibition. In our work, we have investigated the adaptive mechanism of BCL2 inhibition resistance in AML. We have explored this mechanism using an AML cell line model in which immature AML cells continuously generate differentiated AML cells (OCI-AML8227). We used fluorescence-activated cell sorting to isolate immature CD34+/CD38- OCI-AML8227 cells. After 72 hours in liquid culture, we observed that these cells recapitulated a pool of differentiated CD34-/CD38+/CD14+ cells. Furthermore, we used single cell ATAC sequencing to confirm that the unfractionated pool of OCI-AML8227 cells display chromatin accessibility profiles resembling cells across the continuum of myeloid differentiation. To understand whether this model is appropriate to investigate the differentiation-state-specific differences in sensitivity to BLC2 inhibition, we measured the viability of immature CD34+ and differentiated CD34- OCI-AML8227 cells following BCL2 inhibition. Consistent with previous studies, immature CD34+ cells were significantly more sensitive to BCL2 inhibition than differentiated CD34- cells. In addition, BLC2 inhibition of unfractionated OCI-AML8227 cells resulted in an 82% increase in the number of differentiated CD34-/CD38+ cells. While the number of immature CD34+/CD38- cells was reduced following BCL2 inhibition, it was only reduced by 27%. Furthermore, there was no change in the apoptosis marker Annexin V among immature CD34+/CD38- cells. These results support an adaptive resistance model where AML cells acquire resistance through differentiation state plasticity. We will explore this mechanism by following the temporal dynamics of immature and differentiated blasts following BCL2 inhibition. Using single cell barcoding, we will not only be able to trace the trajectories of these populations but also investigate transcriptional adaptation of the immature and differentiated populations. With an understanding of the adaptive mechanism of resistance to BCL2 inhibition, we can nominate alternative targets to prevent the outgrowth of resistance populations and design more effective, rational BLC2-inhibitor-based therapeutic strategies.

Characterizing Inferred Functional and Phenotypical States of Newly Diagnosed Acute Myeloid Leukemia from Single Cell Transcriptomic Profiling

Background: Acute myeloid leukemia (AML) is a heterogeneous disease, marked by leukemia stem cells (LSCs), which significantly contribute to treatment resistance and dismal prognosis. While appreciable efforts have been made to study the diversity of functional and phenotypic states of LSCs, integrating these states from inferred single cell profiling has not been explored. Moreover, the spatial distribution and variability of hierarchical leukemic states within diverse cytogenetic groups, and their potential connection to transcriptional profiles, remain understudied, highlighting a gap in our understanding of this complex disease. Methods: To investigate the heterogeneity across different cytogenetics,we performed single cell RNA profiling (scRNA) of bone marrow (BM) mononulear cells from 20 newly diagnosed adult AML patients with diploid (n=7), del5/5q (n=5), del7/7q (n=5) and double deletion (del5/5q & del7/7q; n=3). A total of 111,130 cells passed quality assessment, of which 56,168 (50.5%) cells were identified as AML cells by integrating flow cytometry, immunohistochemistry, copy number variation, and FISH with the scRNA expression profiles. Functional states of AML cells were predicted by a recently proposed hierarchy of AML LSCs representing distinct maturation states (Zeng et al Nat Med 2022). Phenotypical states were annotated by Symphony using a healthy BM and validated in newly sequenced healthy BM samples. We also applied deconvolution scRNA analysis with spatial GeoMx profiling to identify spatial location of primitive versus differentiated AML cells. Results: Differential gene expression and gene set enrichment analysis (GSEA) revealed transcriptional and biological diversity across cytogenetic groups. Del7/7q cells showed high expression of human leukocyte antigen (HLA) genes and CD74, consistent with enrichment for a inflammatory phenotype revealed by GSEA. Del5/5q group demonstrated enrichment in proliferative pathways and heat shock proteins. Some diploid cells showed high expression of monocytic markers, while the others were highly expressing GMP markers, indicating the co-existence of different subclones. Differentiation states of leukemic cells are reflected in the hierarchy of cellular compositions. There is a diverse spectrum of cell types within each cytogenetic group and each patient. However, AML cells from diploid patients were enriched for GMP-like and monocyte-like cells, while phenotypically HSC-like cells showed an incremental trend in the sequence of diploid, del5/5q, del7/7q, and double deletion (Figure 1A). The inferred functional LSC states in our data also showed diverse distribution across cytogenetic groups, with nondiploid cells harboring more LSC. We also evaluated the biological differences of cytogenetic groups within each phenotypical and functional subset. Our data revealed consistent upregulation of proliferation in del5/5q and inflammation in del7/7q with double deletion preserving both features. However, diploid cells were downregulated in almost all. These data suggest cytogenetics as a major driver of ITH even for phenotypically or functionally similar AML cells. By correlating phenotypical axis with LSC states, we found a shared transcriptional program in LSC-like states across phenotypical axis except for Mono-like and DC-like cells, demonstrating the broad distribution of inferred functional LSC spanning the phenotypical axis from primitive to differentiated cells (Figure 1B). This was also observed in other LSC signatures, suggesting that AML cells with LSC capacity exist across the phenotypic of HSC-like cells to more phenotypically differentiated cells. We then performed spatial deconvolution using GeoMx of CD34 + or CD68 + myeloid areas of interest using the phenotypical signature. GeoMx revealed that primitive cells were more likely to be proximal to bone, while more differentiated states were enriched in distal region from the bone, indicating a spatial migration at time of differentiation. Conclusions: ScRNA profiling of newly diagnosed AML revealed the existence of heterogeneous LSC functional states underlying varying degrees of differentiation. We identified cytogenetics as a major source of ITH. Also, primitive cells were likely to localize to bone regions. These findings provide new insights in developing specific therapeutic strategies to improve clinical outcomes.

Single-Cell RNA Sequencing Facilitates Study of Cancer Mechanisms across Pediatric Leukemias

Leukemia is the most common pediatric cancer, diagnosed in over 3,000 children annually in the US. While the prognosis for many pediatric leukemias has significantly improved with the addition of DNA sequencing to the diagnosis and treatment selection process, the molecular mechanisms driving some cases remain inscrutable. In this study, we performed single-cell RNA sequencing on a cohort of pediatric leukemia samples (n = 72) to explore intra- and inter-patient heterogeneity across multiple types of malignancies: acute megakaryoblastic leukemia (AMKL, n = 1), acute myeloid leukemia (AML, n = 14), acute promyelocytic leukemia (APL, n = 1), B-cell acute lymphoblastic leukemia (B-ALL, n = 32), anaplastic large cell lymphoma (ALCL, n = 1), myelodysplastic syndrome (MDS, n = 1), mixed phenotype acute leukemia (MPAL, n = 2), Ph-like B-cell acute lymphoblastic leukemia (Ph-like, n = 8), and T-cell lymphoblastic leukemia (T-ALL, n = 12). Using the 10x Chromium system, we generated single-cell RNA sequencing data for 183,321 cells from cryopreserved blood and bone marrow samples collected from patients at diagnosis (n = 65), remission (n = 3), and/or relapse (n = 4). Sequencing data were aligned to GRCh38 and counted using cellranger-6.0.1, with downstream analysis performed using Seurat 4.0.2 in R 4.0.3. Consistent with prior single-cell studies of leukemia, we observed a high degree of inter-patient heterogeneity: cancer cells from each patient showed distinct transcriptional signatures (Figure A; circled clusters indicate mature cells of each type clustering together, while other clusters correspond to leukemic cells from a single patient). Regardless of leukemia type and cell of origin, many patient samples contained cells most transcriptionally similar to hematopoietic stem cells (HSCs; Figure B). At diagnosis, the proportion of HSCs-like cells ranged from 7% in patients with MPAL to 46% in AML. Samples collected at relapse showed a striking increase in the proportion of HSC-like cells in both B-ALL (97%, compared to 45% at diagnosis) and Ph-like (92%, compared to 16% at diagnosis). Comparative analysis of this cohort additionally revealed marker genes specific to each leukemia, reflecting the diverse cell of origin and molecular mechanisms driving each cancer, including PRDM2 and ID3 for B-ALL, PRDM16 and CSF3R for AML, as well as SKI and PTPRC for MPAL. In this study, we use single-cell RNA sequencing of a large pediatric leukemia cohort to gain novel insights into the transcriptional drivers and heterogeneity of leukemias. Future work will extend these observations to explore transcriptional signatures associated with prognosis, both common and unique to individual cancers. In the long term, the findings from this research will facilitate improved diagnosis, patient stratification, and treatment selection for pediatric patients diagnosed with leukemia.

Identification of Acute Myeloid Leukemia Cell Surface Therapeutic Targets Using Single Cell RNA Sequencing Supported By Surface Proteomics

Background: Acute myeloid leukemia (AML) comprises diverse genomic subgroups and remains hard to treat in most patients. Despite breakthroughs in the therapeutic arsenal in recent years, clinical usage of therapeutic antibodies or chimeric antigen receptor T (CAR-T) cells has been lagging in contrast to other hematological malignancies. In fact, CD33 represents the only antibody-based strategy approved for this disease to date, highlighting the need to identify new promising targets. AML cells span a wide range of aberrant myeloid differentiation programs, complexifying the identification, by bulk genomics, of targets expressed in the most immature leukemic cells. Aims and Methods: To identify the expression landscape of surface proteins in immature leukemic cells, we performed single-cell RNA sequencing (scRNA-seq, 10x 3' Reagent Kits) of primary human AML cells from 20 specimens of the Leucegene cohort enriched in intermediate and adverse genetic backgrounds ( KMT2A-rearranged n=5, chromosome 5 and/or 7 deletions (abn5/7, n=5) complex karyotype (n=4), NPM1/DNMT3A/FLT3-ITD triple-mutant (n=3) and others (n=3)). A Random Forest classifier was developed to unbiasedly classify AML cells into distinct differentiation stages using normal bone marrow-derived scRNA-seq data from the Human Cell Atlas (HCA) consortium. Genes were scored based on their probability of coding for proteins expressed at the cell surface using the SPAT algorithm developed by our group (https://doi.org/10.1101/2023.07.07.547075), retaining high score ones. To validate surface expression, we concomitantly analyzed the surface proteome (hereafter named surfaceome) of 100 primary human AML samples from the Leucegene cohort, including all 20 samples profiled by scRNA-seq. Results: After quality control, we profiled and characterized 103 690 high quality cells (mean of 5185 cells/sample). We trained a Random Forest classifier to annotate cells in a two step process, first identifying plasma cells based on a restricted list of genes abundantly expressed in these cells and subsequently assigning the remaining cells to one of 33 cell types. We performed a five-fold cross validation of the model and subsequently determined the accuracy of our classifier to be 92% on the test subset of the HCA data. Applied to our AML cell collection, a total of 35 053 cells (34%) were unbiasedly classified as Hematopoietic Stem Cell (HSC)-like, corresponding to the most phenotypically immature leukemic cells in each patient sample (ranging from 4 to 74 %). Accordingly, HSC-like AML cells preferentially express genes associated with normal HSCs, such as CD34, FAM30A, and SPINK2, and globally lack expression of mature lineages defining genes, further validating our classifier. The proportion of HSC-like cells varied among AML subgroups, and was lowest in KMT2A-r AML (median 19%) and highest in abn5/7 samples (46%). Integration of our AML atlas using Harmony algorithm preserved differentiation hierarchies across samples, with most cell types, including HSC-like cells, occupying a defined area in the low dimensional embedding. To identify new surface antigens specifically expressed in immature leukemic cells, we compared the high (≥8) SPAT score gene expression profile of AML HSC-like cells with that of normal HSC cells (HCA), and identified 60 genes significantly overexpressed in AML immature cells. Of those, 39 genes were also detected at the protein level by the surfaceome analysis, supporting their predicted expression at the cell surface in AML samples. 59% of these 39 genes (n=23) were detected in over 80% of the specimens analyzed by the surfaceome, and thus are nearly universally expressed in our AML cohort. To identify targets of therapies that could be repurposed, we next evaluated the relevance of our findings by querying the Thera-SAbDab database. Most interestingly, 8 of the 39 AML specific HSC markers are targeted by therapeutic antibodies FDA-approved or in clinical trials for the treatment of AML (n=4, IL3RA, FLT3, CD37 and TNFRSF10B) or other indications (n = 4). Conclusion Our genetically diverse AML single-cell atlas, supported by mass spectrometry, enables the identification of both subset-specific and pan-AML surface protein genes. These represent potential targets for antibody based strategy development or therapy repurposing in AML.

An Assessment of Marrow-Infiltrating T Cells in Early Relapsed Hematologic Cancer Patients Post Allogeneic Hematopoietic Stem Cell Transplantation

Background: Relapse is the major treatment failure of allogeneic hematopoietic stem cell transplantation (HSCT) in patients with hematologic malignancies. Especially, early relapse had seriously bad prognosis. And tumor-infiltrating T cells play an important role in disease progression and recurrence. However, there is lack of data about the marrow-infiltrating T cell of early relapsed patients compared to those with complete remission (CR) at an early timepoint after HSCT. Therefore, we analyzed the differentiation stage and expression of inhibitory receptors of the marrow-infiltrating T cell in the early stages of allogeneic HSCT. Method: A total of 40 samples from patients treated at Chungnam National University Hospital were collected between April 2021 and October 2022. We used flow cytometry to classify T cells into five subtypes using CD45RA, CD95, and CCR7: naïve, stem cell memory (SCM), central memory (CM), effector memory (EM) and effector memory with positive CD45RA (EMRA). Otherwise, we analyzed regulatory T cell (Treg) by defining as CD25 highCD127 low and divided them into three categories using CD25 and CD45RA: naïve, active and non-suppressive Treg. We defined early relapse (ER) as relapse within 6 months of allogeneic HSCT. High-throughput single-cell RNAseq assays from BM lymphocytes isolated by density gradients were performed to determine the expression and cell composition of previously irradiated markers. Results: With this approach, marrow-infiltrating T cells were analyzed in 40 patients (10 patients with early relapse) with a median age of 57 years (ranged from 30 to 71). AML is the most common disease at 60%, followed by MDS (20%), ALL (15%) and PMF (2.5%). Stem cell sources were 32.5% from matched sibling donor, 37.5% from haplo-identical donor and 27.5% from matched unrelated donor. All patients received mobilized peripheral blood stem cell transplantation. Marrow-infiltrating CD3+T cell counts were lower in patients with allogeneic HSCT compared to normal subjects (CD3+T cells: 13.89% in HSCT patient and 9.116% in ER patients vs. 34.19% in normal subjects, p< 0.0001). In addition, CD3+CD8+T cells were higher in CR than ER patients (CD3+CD8+T cells: 50.39% in CR vs. 30.41% in ER patients, p< 0.0033). In HSCT patients, naïve was higher, and EM and EMRA were lower than those in normal subjects among CD4+T cells (naïve; 8.629% in ER patients and 5.119% in CR patient's vs 45.76% in normal subjects, p< 0.0001/EM; 54.04% in ER patients and 55.93% in CR patients vs 18.61 in normal subject, p= 0.0003 and p< 0.0001 respectively). Of CD3+T cells, the proportion and expression level of TIM-3 was higher ER patients than in CR patients (56.33% in ER patients vs 39.20% in CR patients, p= 0.0185/MFI; 1873 in ER patients vs. 826.7 in CR patients, p= 0.0331). Of CD4+T cells, the percentage of TIM-3 was lower in normal subjects than in allogeneic HSCT patients (7.794% in normal vs 30.11% in CR patients and 26.04% in ER patients, p= 0.0002 and p= 0.0102 respectively). In patients with ER, naïve and EM was higher than those in CR patients among CD8+T cells (naïve; 8.680% in ER patients vs 4.066% in CR patients, p= 0.4001 /EM; 54.44% in ER patients vs 44.33% in CR patients, p= 0.3039). Of CD8+ EMRA T cell, the expression of TIM-3 was higher in ER patients than in CR patients (MFI; 1441 in HSCT patients vs 421.7 in normal, p=0.1038). Otherwise, of CD3+CD4-CD8-, The percentage of TIM3 was higher in ER patients than CR patients (80.33 % in ER patients vs 62.49% in CR patients, p= 0.0333). Treg levels were no significant difference between CR and ER. We have obtained single-cell sequencing data from two CR (complete response) patients and two ER (early relapse) patients, representing each group. Since the classified groups exhibited ambiguous phenotypes, they were reclassified based on specific markers of cell groups. The reclassified groups showed a higher proportion of HCS (Hematopoietic Stem Cell)-like cells in relapsed patients (+10.14%, P=0.368) and a lower proportion of CD8+ T cells (-13.40%, p=0.068). The HSC-like cells in relapsed patients exhibited higher expression of TIM3 ligand than in CR patients Conclusion: Among the IRs, TIM3 had the highest expression of all cell groups (CD4 T cells, CD8 T cells and Treg) compared with other IRs at the early stage of allogeneic HSCT. So, TIM3 might be a potential target for preventing relapse in hematologic cancer patients after allogeneic HSCT in the future.

Single-cell transcriptomics reveals multiple chemoresistant properties in leukemic stem and progenitor cells in pediatric AML

BackgroundCancer patients can achieve dramatic responses to chemotherapy yet retain resistant tumor cells, which ultimately results in relapse. Although xenograft model studies have identified several cellular and molecular features that are associated with chemoresistance in acute myeloid leukemia (AML), to what extent AML patients exhibit these properties remains largely unknown.ResultsWe apply single-cell RNA sequencing to paired pre- and post-chemotherapy whole bone marrow samples obtained from 13 pediatric AML patients who had achieved disease remission, and distinguish AML clusters from normal cells based on their unique transcriptomic profiles. Approximately 50% of leukemic stem and progenitor populations actively express leukemia stem cell (LSC) and oxidative phosphorylation (OXPHOS) signatures, respectively. These clusters have a higher chance of tolerating therapy and exhibit an enhanced metabolic program in response to treatment. Interestingly, the transmembrane receptor CD69 is highly expressed in chemoresistant hematopoietic stem cell (HSC)-like populations (named the CD69+ HSC-like subpopulation). Furthermore, overexpression of CD69 results in suppression of the mTOR signaling pathway and promotion of cell quiescence and adhesion in vitro. Finally, the presence of CD69+ HSC-like cells is associated with unfavorable genetic mutations, the persistence of residual tumor cells in chemotherapy, and poor outcomes in independent pediatric and adult public AML cohorts.ConclusionsOur analysis reveals leukemia stem cell and OXPHOS as two major chemoresistant features in human AML patients. CD69 may serve as a potential biomarker in defining a subpopulation of chemoresistant leukemia stem cells. These findings have important implications for targeting residual chemo-surviving AML cells.

Hoxb5 reprogrammes murine multipotent blood progenitors into haematopoietic stem cell-like cells.

ObjectivesThe expression of transcription factor Hoxb5 specifically marks the functional haematopoietic stem cells (HSC) in mice. However, our recent work demonstrated that ectopic expression of Hoxb5 exerted little effect on HSC but could convert B‐cell progenitors into functional T cells in vivo. Thus, cell type‐ and development stage‐specific roles of Hoxb5 in haematopoietic hierarchy await more extensive exploration. In this study, we aim to investigate the effect of Hoxb5 expression in multipotent blood progenitor cells.Materials and MethodsA Mx1cre/RosaLSL‐Hoxb5‐EGFP/+ mouse model was used to evaluate the effect of Hoxb5 expression in blood multipotent progenitor cells (MPP). Golden standard serial transplantation experiments were used to test the long‐term haematopoiesis potential of Hoxb5‐expressing MPP. Single‐cell RNA‐seq analysis was used to characterize the gained molecular features of Hoxb5‐expressing MPP and to compare with the global transcriptome features of natural adult HSC and fetal liver HSC (FL HSC).ResultsHere, with a mouse strain engineered with conditional expression of Hoxb5, we unveiled that induced expression of Hoxb5 in MPP led to the generation of a de novo Sca1+c‐kit+CD11b+CD48+ (CD11b+CD48+SK) cell type, which can repopulate long‐term multilineage haematopoiesis in serial transplantations. RNA‐seq analysis showed that CD11b+CD48+SK cells exhibited acquired features of DNA replication and cell division.ConclusionsOur current study uncovers that Hoxb5 can empower MPP with self‐renewal ability and indicates an alternative approach for generating HSC‐like cells in vivo from blood lineage cells.

Potential Use of Patient-Specific Induced Pluripotent Stem Cell for Liver Fibrosis Thalassemia Treatment Management

Thalassemia is the most common inherited single gene blood disease worldwide and present a significant health problem in the world. Approximately, 1.5% of the global populations (An estimated 80–90 million people) are carriers of β-thalassemia. Around 5% of Indonesia population is thought to carry the thalassemia gene. The globin imbalance in β-thalassemia major causes hemolysis and ineffective erythropoiesis which results in anemia leading to increases of iron absorption. Furthermore, repeated blood transfusion and long-term increased iron absorption will lead to excessive accumulation of iron in vital organs, especially in the liver, causes liver fibrosis then leading to liver disease. Iron overload can be controlled by iron chelating drugs with the risk of side effects; therefore, a breakthrough is needed. Stem cell technology has a potential to provide novel insight in thalassemia major, through induced pluripotent stem cells (iPSCs) who has the ability to differentiate into hepatic stellate cells (HSCs)-like cells. iPSCs derived HSC-like cells (iPSC-HSCs) present the phenotypic and functional characteristics of HSCs. The utilization of iPSCs is a new option in personalized thalassemia management.

Engineering of Induced Pluripotent Stem Cell-Derived Hemogenic Endothelium

Background: Autologous cellular therapy of hematological malignancies and primary immunodeficiencies has been limited by the inability to produce functional hematopoietic stem cells (HSCs) from patient-derived induced pluripotent stem cells (iPS). Previous approaches have failed to demonstrate multi-lineage potential or secondary bone marrow engraftment in murine hosts - the gold standards for demonstrating HSC self-renewal and differentiation. Building upon recent evidence that HSCs derive from the definitive hemogenic endothelium (HE) of the embryonic lateral plate mesoderm, we set out to engineer iPS-derived HSCs by directed differentiation, and to investigate potential applications for modeling genetic blood diseases in vitro .Methods: Following embryoid body formation, human iPS derived from bone marrow mesenchymal stem cells underwent stepwise, morphogen-directed differentiation into HE, followed by polycistronic lentiviral transduction with five transcription factors (RUNX1, ERG, LCOR, HOXA5, HOXA9) on day 3 of endothelial-to-hematopoietic transition (EHT) induction, to enable further conversion to HSC-like cells with lymphoid potential in vitro and known multi-lineage engraftment capacity in vivo . A candidate screen of 17 cytokines, epigenetic modifiers and small molecules aimed at inducing HE proliferation was conducted during EHT induction in a RUNX1c gene reporter assay. To model human disease, dermal fibroblast-derived iPS from a hypomorphic RAG2 Omenn syndrome patient were differentiated into HSCs, and cocultured with MS5 or OP9-DL1 stromal cells to induce B or T cell development. Fluorescence-activated cell sorting (FACS) was performed weekly for 6 weeks after initiating lymphoid differentiation.Results: In the candidate screen, IFN-γ enhanced RUNX1c reporter 2.4-fold during EHT, which was confirmed by flow cytometry showing increased hematopoietic progenitor cells (CD34+CD45+). Several compounds were suppressive to EHT, including IFN-α, which abrogated EHT enhancement when combined with IFN-γ. Despite 1 week of lymphoid-directed differentiation, cell populations derived from mutant RAG2 iPS cells showed a predominance of myeloid progenitors (CD38+CD45+CD45RA+), with only 5% of progenitors manifesting a lymphoid phenotype (CD10+CD34+CD45RA+). In contrast, wildtype iPS-derived HSCs produced CD19+IgM- B cell progenitors as well as mature IgM+ B cells (8% versus .06% of CD19+CD21+ B cells), which were comparable to cord blood controls; CD3+CD4+CD8+ T cells were also identified after 3 weeks. Ultimately, we determined that RAG2 -mutant iPS were unable to generate CD19+ B cells and arrested at the early T cell stage (CD5+CD4-CD8-).Conclusion: We determined that transient exposure to IFN-γ greatly improved the EHT of HE, which may bolster the typically low yield of iPSC-derived HSCs. In a lymphoid differentiation assay, wildtype iPS-derived HE were comparable to CB in the formation of mature and immature B/T cells, whereas RAG2 -derived HEs arrested at the pro-thymocyte stage. This finding suggests that our approach to creating engraftable HSCs holds significant potential for modeling hematopoietic disease, both in animal models and as a “disease-on-a-dish,” and for developing therapeutic strategies in genetic blood disorders. DisclosuresNo relevant conflicts of interest to declare.

Enhanced HSC-like cell generation from mouse pluripotent stem cells in a 3D induction system cocultured with stromal cells

BackgroundDecades of efforts have attempted to differentiate the pluripotent stem cells (PSCs) into truly functional hematopoietic stem cells (HSCs), yet the problems of low differentiation efficiency in vitro and poor hematopoiesis reconstitution in vivo still exist, mainly attributing to the lack of solid, reproduced, or pursued differentiation system.MethodsIn this study, we established an in vitro differentiation system yielding in vivo hematopoietic reconstitution hematopoietic cells from mouse PSCs through a 3D induction system followed by coculture with OP9 stromal cells. The in vivo hematopoietic reconstitution potential of c-kit+ cells derived from the mouse PSCs was evaluated via m-NSG transplantation assay. Flow cytometry analysis, RNA-seq, and cell cycle analysis were used to detect the in vitro hematopoietic ability of endothelial protein C receptor (EPCR, CD201) cells generated in our induction system.ResultsThe c-kit+ cells from 3D self-assembling peptide induction system followed by the OP9 coculture system possessed apparently superiority in terms of in vivo repopulating activity than that of 3D induction system followed by the 0.1% gelatin culture. We interestingly found that our 3D+OP9 system enriched a higher percentage of CD201+c-kit+cells that showed more similar HSC-like features such as transcriptome level and CFU formation ability than CD201-c-kit+cells, which have not been reported in the field of mouse PSCs hematopoietic differentiation. Moreover, CD201+ hematopoietic cells remained in a relatively slow cycling state, consistent with high expression levels of P57 and Ccng2. Further, we innovatively demonstrated that notch signaling pathway is responsible for in vitro CD201+ hematopoietic cell induction from mouse PSCs.ConclusionsAltogether, our findings lay a foundation for improving the efficiency of hematopoietic differentiation and generating in vivo functional HSC-like cells from mouse PSCs for clinical application.

Directed differentiation of human induced pluripotent stem cells to hepatic stellate cells.

Hepatic stellate cells (HSCs) are nonparenchymal liver cells responsible for extracellular matrix homeostasis and are the main cells involved in the development of liver fibrosis following injury. The lack of reliable sources of HSCs has hence limited the development of complex in vitro systems to model liver diseases and toxicity. Here we describe a protocol to differentiate human induced pluripotent stem cells (iPSCs) into hepatic stellate cells (iPSC-HSCs). The protocol is based on the addition of several growth factors important for liver development sequentially over 12 d. iPSC-HSCs present phenotypic and functional characteristics of primary HSCs and can be expanded or frozen and used to perform high-throughput in vitro studies. We also describe how to coculture iPSC-HSCs with hepatocytes, which self-assemble into three-dimensional (3D) hepatic spheroids. This protocol enables the generation of HSC-like cells for in vitro modeling and drug screening studies.

Single-cell analyses and machine learning define hematopoietic progenitor and HSC-like cells derived from human PSCs

AbstractHematopoietic stem and progenitor cells (HSPCs) develop in distinct waves at various anatomical sites during embryonic development. The in vitro differentiation of human pluripotent stem cells (hPSCs) recapitulates some of these processes; however, it has proven difficult to generate functional hematopoietic stem cells (HSCs). To define the dynamics and heterogeneity of HSPCs that can be generated in vitro from hPSCs, we explored single-cell RNA sequencing (scRNAseq) in combination with single-cell protein expression analysis. Bioinformatics analyses and functional validation defined the transcriptomes of naïve progenitors and erythroid-, megakaryocyte-, and leukocyte-committed progenitors, and we identified CD44, CD326, ICAM2/CD9, and CD18, respectively, as markers of these progenitors. Using an artificial neural network that we trained on scRNAseq derived from human fetal liver, we identified a wide range of hPSC-derived HSPCs phenotypes, including a small group classified as HSCs. This transient HSC-like population decreased as differentiation proceeded, and was completely missing in the data set that had been generated using cells selected on the basis of CD43 expression. By comparing the single-cell transcriptome of in vitro–generated HSC-like cells with those generated within the fetal liver, we identified transcription factors and molecular pathways that can be explored in the future to improve the in vitro production of HSCs.

3074 – SINGLE CELL TRANSCRIPTOMIC ANALYSIS COMBINED WITH MACHINE LEARNING IDENTIFIES HSC-LIKE CELLS DIFFERENTIATED IN VITRO FROM HUMAN IPSCS.

During embryonic development, haematopoietic stem and progenitor cells (HSPCs) develop in different waves at various anatomical sites. This process can be recapitulated in vitro by cytokine-mediated differentiation of human pluripotent stem cells (hPSCs) although the differentiation of haematopoietic stem cells (HSCs) remains a major challenge in the field. To depict the developmental dynamics and to study the heterogeneity of HSPCs that can be generated in vitro from hPSCs, we employed single cell RNA sequencing (scRNAseq) and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq). We identified the transcriptome and functionally validated naïve progenitors as well as erythroid, megakaryocyte and leukocyte-committed progenitors and we associated to these the expression of CD44, CD326, ICAM2/CD9 and CD18, respectively. To identify and compare the in vitro-generated cell types to those developed in vivo, we employed a machine learning approach. We trained an artificial neural network (ANN) using a human fetal liver scRNAseq dataset of hematopoietic cells from HSCs to mature cell types. We identified a wide range of hPSCs-derived phenotypes, including a small group classified as HSC/MPP. This transient HSC-like population decreased with the progression of the differentiation protocol and was not identified by the ANN in the datasets that we generated from sorted CD43+ cells. By comparing the transcriptome of these in vitro-generated HSC/MPP-like cells with those within the fetal liver we identified transcription factors and molecular pathways that can be exploited to improve the in vitro production of HSCs in the future. Finally, when these HSC-like cells were compared to yolk sac and AGM cells we observed that in vitro derived progenitors displayed signatures of high oxidative respiration, suggesting an overall divergent metabolic profile.

1015 – HUMAN HAEMATOPOIETIC STEM AND PROGENITOR CELL LANDSCAPES: LOCATION MATTERS

In adults, most hematopoietic stem and progenitor cells (HSPCs) reside within the bone marrow (BM), giving rise to all mature blood cells. Yet at any given time, a small proportion of HSPCs circulates in peripheral blood (PB), and under severe stress and disease, the spleen can significantly contribute to blood production. However, the cellular, molecular and functional composition of circulating and extramedullary HSPC pools remains unexplored. Here I will discuss the single cell characterisation of the adult human HSPC pool found in spleen and non-mobilised PB, comparing and contrasting it to BM. Using matched and unmatched samples from deceased and living donors, we profiled more than 50,000 single CD34+ HSPCs by scRNA-seq and 3,900 single phenotypic haematopoietic stem cells / multipotent progenitors (HSC/MPPs) in functional assays. In BM, we find a topography of the hematopoietic hierarchy that supports continuous HSPC proliferation and blood production. In contrast, the cellular configuration in extramedullary tissues is positioned for lineage-primed demand-adapted haematopoiesis, including a molecularly distinct subset of HSC/MPPs not found in BM. We also find steady-state non-mobilised PB is dominated by quiescent HSC-like cells and non-proliferative early progenitors functionally restricted to erythrocyte and megakaryocyte production. PB HSC/MPPs thus sustain a unique differentiation potential and configuration in healthy conditions, but which become imbalanced with age and in haematological conditions. Overall these data identify extramedullary cellular reservoirs for demand-adapted haematopoiesis and provide a framework of clinical relevance.

A bioengineering approach to Schlemm's canal-like stem cell differentiation for in vitro glaucoma drug screening

Human Schlemm's canal (HSC) cells are critical for understanding outflow physiology and glaucoma etiology. However, primary donor cells frequently used in research are difficult to isolate. HSC cells exhibit both vascular and lymphatic markers. Human adipose-derived stem cells (ADSCs) represent a potential source of HSC due to their capacity to differentiate into both vascular and lymphatic endothelial cells, via VEGF-A and VEGF-C. Shear stress plays a critical role in maintaining HSC integrity, function, and PROX1 expression. Additionally, the human trabecular meshwork (HTM) microenvironment could provide cues for HSC-like differentiation. We hypothesize that subjecting ADSCs to VEGF-A or VEGF-C, shear stress, and co-culture with HTM cells could provide biological, mechanical, and cellular cues necessary for HSC-like differentiation. To test this hypothesis, effects of VEGF-A, VEGF-C, and shear stress on ADSC differentiation were examined and compared to primary HSC cells in terms of cell morphology, and HSC marker expression using qPCR, immunoblotting, and immunocytochemistry analysis. Furthermore, the effect of co-culture with HTM cells on porous scaffolds on ADSC differentiation was studied. Treatment with VEGF-C under shear stress is effective in differentiating ADSCs into PROX1-expressing HSC-like cells. Co-culture with HTM cells on porous scaffolds leads to HTM/ADSC-derived HSC-like constructs that regulate through-flow and respond as expected to dexamethasone. Statement of significanceWe successfully generated human Schlemm's canal (HSC) like cells from adipocyte-derived stem cells induced by biochemical and biomechanical cues as well as bioengineered human trabecular meshwork (HTM) on micropatterned, porous SU8 scaffolds. These stem cell-derived HSC-like cells co-cultured with HTM cells on SU8 scaffolds can regulate through-flow, and in particular, are responsive to steroid treatment as expected. These findings show that ADSC-derived HSC-like cells have the potential to recreate the ocular outflow pathway for in vitro glaucoma drug screening.To the best of our knowledge, it is the very first time to demonstrate derivation of Schlemm's canal-like cells from stem cells. It provides an important alternative source to primary Schlemm's canal cells that are very difficult to be isolated and cultured from human donors.

Synergy of NUP98-HOXA10 Fusion Gene and NrasG12D Mutation Preserves the Stemness of Hematopoietic Stem Cells on Culture Condition.

Natural hematopoietic stem cells (HSC) are susceptible and tend to lose stemness, differentiate, or die on culture condition in vitro, which adds technical challenge for maintaining bona fide HSC-like cells, if ever generated, in protocol screening from pluripotent stem cells. It remains largely unknown whether gene-editing of endogenous genes can genetically empower HSC to endure the culture stress and preserve stemness. In this study, we revealed that both NUP98-HOXA10HD fusion and endogenous Nras mutation modifications (NrasG12D) promoted the engraftment competitiveness of HSC. Furthermore, the synergy of these two genetic modifications endowed HSC with super competitiveness in vivo. Strikingly, single NAV-HSC successfully maintained its stemness and showed robust multi-lineage engraftments after undergoing the in vitro culture. Mechanistically, NUP98-HOXA10HD fusion and NrasG12D mutation distinctly altered multiple pathways involving the cell cycle, cell division, and DNA replication, and distinctly regulated stemness-related genes including Hoxa9, Prdm16, Hoxb4, Trim27, and Smarcc1 in the context of HSC. Thus, we develop a super-sensitive transgenic model reporting the existence of HSC at the single cell level on culture condition, which could be beneficial for protocol screening of bona fide HSC regeneration from pluripotent stem cells in vitro.

Modulation of Mesodermal Patterning Combined with High VEGFA Concentrations Promote Robust Arterial Hemogenic Endothelium Differentiation from Human iPSCs

One of the most promising objectives of clinical hematology is to derive transplantable autologous hematopoietic stem/progenitor cells (HSPCs) from human iPSCs ex vivo, but efficient and clinically relevant methodologies remain unavailable. Observations in the developing embryo indicate that definitive HSPCs arise in the dorsal aorta from hemogenic endothelium (HE) in close association with an arterial vascular endothelial niche. The Notch pathway plays a key role in arterial and HSC differentiation; in the embryo, only HE populations found in arterial regions with active Notch signaling through Delta-like ligand 4 (Dll4) and Jagged 1 (Jag1) lead to HSPCs with repopulating potential. Recent studies have also shown that modulation of mesodermal patterning through repression and activation of Activin/Nodal and Wnt/β-catenin pathways, respectively, promotes arterial programs and definitive hematopoiesis. To facilitate the development of functional HSPCs from human iPSCs, we previously developed a simple, monolayer-based, chemically-defined, and scalable differentiation protocol requiring no replating or embryoid body (EB) formation (commercially available as STEMdiffTM Hematopoietic Kit, Stem Cell Technologies). During the first 3 days, mesodermal specification is induced using morphogens (bFGF, BMP4, VEGF 10ng/mL) and, for the subsequent 18 days, cells are further differentiated into HSPCs with the addition of hematopoietic cytokines (SCF, Flt3L and TPO). As previously presented by our group, this differentiation system recapitulates the successive waves of hematopoiesis during development and leads to robust production of immunophenotypic HSC-like cells (CD34+CD38-CD90+CD45RA-CD49f+). However, these cells do not result in efficient, long-term engraftment in immunodeficient (NSG) mouse models. Characterization of the supportive monolayer from which HSPCs arise during vitro differentiation revealed limited percentages of arterial HE (CD43-CD45-CD34hiCD144+CD73-Dll4+) and arterial endothelium (CD43-CD45-CD34hiCD144+CD73midCD184+), and overabundance of stromal cells (CD43-CD45-CD34-CD144-) which upregulate MSC markers CD105, CD73, and CD90 at later stages in culture. This provides a possible explanation for the lack of engraftment potential of iPSC-derived HSPCs in this system (Panel A). To restrict stromal development and further promote differentiation and maintenance of a supportive arterial endothelial niche, we modified the standard differentiation protocol by addition of CHIR99021 (CHIR) and SB431542 (SB) during the mesodermal stage of differentiation (days 2-3) to activate Wnt/β-catenin and block of Activin/Nodal signaling, respectively. Given that VEGF acts upstream of the Notch pathway during arterial endothelial differentiation, we also increased the concentration of VEGFA 20-fold throughout differentiation (200ng/mL). Our results showed that mesodermal patterning alone (CHIR/SB) activated critical HoxA cluster genes in both early endothelial and late HSPC populations but was insufficient to repress stromal production and maintain an endothelial niche beyond early culture days. However, increased VEGF concentrations, alone or in combination with CHIR/SB, markedly reduced stromal differentiation (Panel B) and enhanced arterial endothelium formation (Panel C) compared to the standard system (control). Importantly, combination treatments also led to significantly higher percentages of arterial HE at days 5 and 7 (panel D). Current assessment of these treatments on the hematopoietic potential of the system is ongoing, and include NSG mouse transplantations. Overall, our data indicate that commercially available technologies can be further modified and improved to move closer to chemically-defined and scalable HSPC differentiation protocols. DisclosuresLarochelle:Stem Cell Technologies: Patents & Royalties: StemDiff Hematopoietic Differentiation Kit.

Generation of Hepatic Stellate Cells from Human Pluripotent Stem Cells Enables In Vitro Modeling of Liver Fibrosis

The development of complex invitro hepatic systems and artificial liver devices has been hampered by the lack of reliable sources for relevant cell types, such as hepatic stellate cells (HSCs). Here we reportefficient differentiation of human pluripotent stem cells into HSC-like cells (iPSC-HSCs). iPSC-HSCsclosely resemble primary human HSCs at the transcriptional, cellular, and functional levels and possess a gene expression profile intermediate between that of quiescent and activated HSCs. Functional analyses revealed that iPSC-HSCs accumulate retinyl esters in lipid droplets and are activated in response to mediators of wound healing, similar to their invivocounterparts. When maintained as 3D spheroids withHepaRG hepatocytes, iPSC-HSCs exhibit a quiescent phenotype but mount a fibrogenic response and secrete pro-collagen in response to known stimuli and hepatocyte toxicity. Thus, this protocol provides a robust invitro system for studying HSC development, modeling liver fibrosis, and drug toxicity screening.