Single Cell Analysis of Adult Human Hematopoietic Stem and Progenitor Cells Identifies a Novel Lymphoid Primed Multipotent Progenitor That Expands in Relapsed Acute Myeloid Leukemia

Background: Hematopoietic stem and progenitor cells (HSPCs) are capable of generating all human blood cells. Although these cells have been extensively evaluated using both sorted (Corces et al. Nat. Genet. 2016) and single cell assays (Pellin et al. Nat. Commun. 2019), there remains significant uncertainty as to the degree of heterogeneity within HSPC subpopulations and their relevance to disease. The phenotypic diversity within HSPCs needs to be better characterized in order to understand the pathogenesis of numerous blood disorders including hematologic malignancies. To address this need, we extensively characterized healthy bone marrow mononuclear cells (BMMCs) with both whole transcriptome analysis (WTA) and surface marker evaluation. We hypothesized that by utilizing concurrent RNA and multiplexed (n=35) surface marker analysis, we could not only improve HSPC clustering but also characterize specific phenotypic states along unique hematopoietic differentiation trajectories. This analysis allowed us to identify new subpopulations within currently defined hematopoietic stem cells (HSCs), multipotent progenitors (MPPs) and lymphoid primed multipotent progenitors (LMPPs). Methods: Three healthy adult BMMC samples were stained with antibody conjugated oligonucleotides (BD Abseq) and analyzed using a nanowell cell capture system (BD Rhapsody). We filtered 8,070 high quality cells for 2,508 HSPCs, myeloid cells, and lymphocyte precursors. These cells were first clustered using matrix factorization in order to identify unique HSPC states. Novel clusters within the HSC, MPP, and LMPP subpopulations were purified using flow cytometry, and functionally evaluated with both in vitro and in vivo assays. Results: The antibody-derived tags (ADTs) obtained from BD Abseq comprised 33 of the most informative features (n=2000) and resulted in more stable clustering as determined by within sum of squares (WSS = 898 versus 934 for mRNA alone for 20 clusters). Additionally, we were able to design a targeted HSPC panel (n=500 genes) with Abseq which identified similar cell clusters compared to the WTA alone and WTA plus ADT data (Rand index = 0.88). HSPC clustering identified the putative hematopoietic stem cell (HSC), a multipotent progenitor (MPP), and a new lymphoid primed multipotent progenitor (LMPP). A new computational sorting strategy was derived to purify these primitive HSPCs, and subsequently validated with flow cytometry. The functional evaluation of these sorted populations revealed that HSC and MPP cells were capable of increased serial replating ability in vitro suggesting enhanced self-renewal capabilities compared to LMPP cells. All three HSPC subpopulations produced gradients of erythroid and myeloid colonies in methylcellulose, and T-cells in T-cell expansion assays in vitro. The HSCs were the only cells that were able to produce long term engraftment in immunodeficient mice after serial transplantations. Additionally, common myeloid progenitor (CMP), granulocyte-monocyte progenitor (GMP), and megakaryocyte-erythroid progenitor (MEP) clusters that expressed canonical surface markers were identified. The primitive HSPC clusters were converted into a signature matrix using CIBERSORTx (Newman et al. Nat. Biotechnol. 2019), and bulk acute myeloid leukemia (AML) and healthy samples were deconvolved into respective healthy cell clusters. Using multivariate Cox proportional hazard analysis, we found that high levels of MPPs at diagnosis was the strongest feature associated with worse overall survival (HR = 78.46, 95% confidence interval 7.32-828). Interestingly, after performing paired analysis of diagnostic and relapse cases, the HSC and LMPP expanded significantly in relapsed AML after chemotherapy (p < 0.05) whereas the MPP expanded considerably after stem cell transplant but did not reach significance (p = 0.09). Conclusions: In summary, we identified new cell type clusters within previously defined HSC, MPP, and LMPP subpopulations, and unique surface marker associations using combined single cell WTA and surface marker analysis. We were able to purify these primitive HSPCs using these new markers, in addition to classical markers like CD34, CD90, and CD45RA. Importantly, deconvolution analysis provided preliminary insights into their clinical relevance in AML. Disclosures Ediriwickrema: Nanosive SAS: Patents & Royalties. Majeti: BeyondSpring Inc: Membership on an entity's Board of Directors or advisory committees; Circbio inc: Membership on an entity's Board of Directors or advisory committees; Kodikaz Therapeutics Solutions Inc: Membership on an entity's Board of Directors or advisory committees; Coherus Biosciences: Membership on an entity's Board of Directors or advisory committees; Acuta Capital Partners: Consultancy; Gilead Sciences, Inc: Patents & Royalties.

Human mesenchymal stem cells inhibit the differentiation and effector functions of monocytes

Although monocytes represent an essential part of the host defence system, their accumulation and prolonged stimulation could be detrimental and may aggravate chronic inflammatory diseases. The present study has explored the less-understood immunomodulatory effects of mesenchymal stem cells on monocyte functions. Isolated purified human monocytes were co-cultured with human umbilical cord-derived mesenchymal stem cells under appropriate culture conditions to assess monocytes’ vital functions. Based on the surface marker analysis, mesenchymal stem cells halted monocyte differentiation into dendritic cells and macrophages and reduced their phagocytosis functions, which rendered an inability to stimulate T-cell proliferation. The present study confers that mesenchymal stem cells exerted potent immunosuppressive activity on monocyte functions such as differentiation, phagocytosis and Ag presentation; hence, they promise a potential therapeutic role in down-regulating the unwanted monocyte-mediated immune responses in the context of chronic inflammatory diseases.

Multiplex Label Free Characterization of Cancer Cell Lines Using Surface Plasmon Resonance Imaging

Rapid multiplex cell surface marker analysis can expedite investigations in which large number of antigens need to be analyzed. Simultaneous analysis of multiple surface antigens at the same level of sensitivity is however limited in the current golden standard analysis method, flow cytometry. In this paper we introduce a surface plasmon resonance imaging (SPRi)-based technique for 44-plex parameter analysis using a single sample, in less than 20 min. We analyzed the expression on cells from five different cancer cell lines by SPRi on a 44-plex antibody array including 4 negative controls and compared the output with flow cytometry. The combined correlation of the markers that showed expression by flow cytometry was 0.76. The results demonstrate as a proof of principle that SPRi can be applied for rapid semi-quantitative multiplex cell surface marker analysis.

HSA+ immature cardiomyocytes persist in the adult heart and expand after ischemic injury

The assessment of the regenerative capacity of the heart has been compromised by the lack of surface signatures to characterize cardiomyocytes. Here, combined multiparametric surface marker analysis with single cell transcriptional profiling and in vivo transplantation, identify the main fetal cardiac populations and their progenitors. We found that cardiomyocytes at different stages of differentiation co-exist during development. We identified a population of immature HSA/CD24+ cardiomyocytes that persists throughout life and that, unlike other cardiomyocyte subsets, actively proliferates up to one week of age and engraft cardiac tissue upon transplantation. In adult heart HSA/CD24+ cardiomyocytes appear as mononucleated cells that cycle and increase in frequency after infarction. Our work identified cell surface signatures that allow the prospective isolation of cardiomyocytes at any developmental stage and the detection of adult cardiomyocytes poised for activation in response to ischemic stimuli. This work opens new perspectives in the understanding and treatment of heart pathologies.

Tenogenic differentiation of mesenchymal stem cells

Category: Basic Sciences/Biologics Introduction/Purpose: Tendon repair has been a challenging issue for surgeons in treating. Although tissue engineering with mesenchymal stem cells (MSC) have been used for tendon repair in both in vivo and in vitro, the stem cells are obtained through invasive procedures, and there is usually a lack of adequate numbers for clinical use. The purpose of this study was to compare the potential of tri-lineage differentiation and to investigate the potential of tenogenic differentiation of human tonsil derived MSCs (T-MSCs), bone marrow derived MSCs (BM-MSCs), and adipose tissue derived MSCs (AD-MSCs). Methods: Each tissue was obtained from 8 patients. After isolation of MSCs, flow cytometry analysis was used to characterize the phenotypes of the MSCs. Differentiation capacity to adipo-, osteo-, and chondrocytes were induced by culturing each MSCs for 3 weeks in commercially available media. Each MSCs was treated with 5ng/ml and 10ng/ml of TGF-ß3 with vehicle control. Results: Immunophenotypic surface marker analysis of BM-MSC, AD-MSC, and TMSCs revealed that these MSCs expressed a typical MSCs. mRNA expression levels of the markers for tri-lineage differentiation were significantly lower in TMSC than other MSCs. The tenogenic transcription factor, scleraxis, showed a statistically significant increase in all MSCs differentiation groups except for the 7th day TMSC differentiation group (Figure). Gene expression of tenascin-C, an ECM glycoprotein, was specifically expressed in the T-MSC differentiation group at 14 days (Figure). Comparing the ratio of collagen 1 to collagen 3 genes, the BM-MSC showed a decrease in the ratio on days 3 and 7 unlike AD-MSCs and TMSCs. Only TMSC showed a significant increase in the ratio compared with other MSCs on the 14th day. Conclusion: The tonsil-MSC has low fat, bone and cartilage differentiation potential and has excellent tendon-specific differentiation potential, thus being highly useful as a tendon-tailored cell therapy agent.

Abstract T P90: Characterization of Inflammatory Cells after EDAS Surgery in Patients with Intracranial Arterial Stenosis

Introduction: Indirect revascularization via encephaloduroarteriosynangiosis (EDAS) establishes collateral flow through new vessels formed from external carotid branches in patients with moyamoya disease and intracranial arterial stenosis (IAS) of non-moyamoya origin. A better understanding of the process of neovascularization is key to improve the potential therapeutic effects of these techniques. To gain a more in-depth understanding on the mechanisms behind the pro-angiogenic effects, we performed detailed evaluation of cellular changes in circulating monocytes. Methods and Results: Dynamics in inflammatory cell were assessed by cell surface marker analysis by flow cytometry of blood from patients at 24, 48 and 72 hours and compared to baseline control (pre-operative) levels from the same patient. While increase in CD45+ cells was noted at 24hrs in all patients, a particularly impressive elevation in the percentage of CD11b/Gr1 positive cells was found in patients at 48 and 72hrs post-surgery. Although CD11b suggested a monocytic population, these cells were F480 low/negative indicating a shift in the differentiation of monocytes or exit of a different population of inflammatory cells from the bone marrow. Conclusions: Our findings are in accordance with recent publications suggesting a pro-angiogenic role of macrophages during vascular expansion and repair. The findings are consistent with the concept that CD11b/Gr1 cells might constitute an important subset of monocyte/macrophages poised to mediate vascular repair upon stroke and could represent a clinically valuable tool.

Bone marrow-derived endothelial progenitor cells confer renal protection in a murine chronic renal failure model

Endothelial cell damage and impaired angiogenesis substantially contribute to the progression of chronic renal failure (CRF). The effect of endothelial progenitor cell (EPC) treatment on the progression of CRF is yet to be determined. We performed 5/6 nephrectomy to induce CRF in C57BL/6 mice. EPCs were isolated from bone marrow, grown in conditioned medium, and characterized with surface marker analysis. The serial changes in kidney function and histological features were scrutinized in CRF mice and EPC-treated CRF (EPC-CRF) mice. Adoptively transferred EPCs were present at the glomeruli and the tubulointerstitial area until week 8 after transfer. In CRF mice, renal function deteriorated steadily over time, whereas the EPC-CRF group showed less deterioration of renal function as well as reduced proteinuria along with a relatively preserved kidney structure. Renal expression of proinflammatory cytokines and adhesion molecules was already decreased in the EPC-CRF group at the early stage of disease, at which point the renal function and histology of CRF and EPC-CRF mice were not different. Angiogenic molecules including VEGF, KDR, and thrombospondin-1, which were decreased in the CRF group, were restored by EPC treatment. In conclusion, EPCs trafficked into the injured kidney protected the kidney from the inflammatory condition and consequently resulted in functional and structural renal preservation. Our study suggests EPCs as a potential candidate for a novel therapeutic approach in CRF.