ALBERT

Description: Intro: In the mouse, hematopoietic stem cells (HSCs) can be isolated and characterized at single cell resolution using a well-defined panel of markers. While it is possible to enrich for human HSCs using a panel of associated markers, similar resolution has not been attained. By profiling HSCs residing in the human fetal liver (FL) using a novel technique called CITE-Seq that combines single cell RNA sequencing (scRNAseq) and cell surface marker interrogation using oligo-tagged antibodies, we aimed to establish an accurate molecular signature of engraftable human HSCs shortly after they arise in development. As HSCs are defined functionally, we have coupled this transcriptomic and protein-level characterization with transplantation assays in immunocompromised NOD scid gamma (NSG) mice to connect expression profiles of cell subsets with functional engraftment. Methods: CITE-Seq was performed on human FL cells (week 19) that showed robust engraftment capability in NSG mice. CD34+ and CD34- cells were magnetically separated and stained with a panel of 19 oligo-tagged antibodies that were deemed relevant to characterize HSCs, including classical HSC markers but also novel targets that were identified in a previous pilot scRNAseq experiment conducted on CD34+ FL cells. From the CD34+ fraction, we sorted live-gated cells (CD34+bulk) as well as a population of cells that was further enriched based on the expression of GPI-80, a marker tightly linked to engraftment potential (CD34+GPI-80+, ~3%). CD34-GlycophorinA(GYPA)- cells were also sorted to assay for the presence of CD34- HSCs. These fractions were then loaded onto the 10x Genomics platform for capture of single cells and subsequent reverse transcription and amplification of both mRNAs and antibody-derived tags (ADTs). Results: Both mRNA and ADT libraries were successfully sequenced, yielding 29-43,000 reads/cell for the mRNA portion and 〉1,500 reads/cell for the ADT fraction. After quality control and filtering, this effort resulted in 8,775 CD34+bulk cells, 7,279 CD34+GPI-80+ cells, and 6,937 CD34-GYPA- cells available for further analysis. Simultaneous transplantation experiments of the fractions assayed by CITE-seq revealed superior engraftment potential of the CD34+GPI-80+ fraction, confirming enrichment for bona fide HSCs at the functional level. This was also reflected in the scRNAseq data where we found enrichment for known HSC markers such as VNN2 (GPI-80), PROM1 (CD133), PROCR (EPCR), THY1 (CD90), ITGA6 (CD49f), HMGA2, CLEC9A and HLF in the CD34+GPI-80+ fraction compared to CD34+bulk cells. As our pilot studies revealed considerable differences in transcriptional expression (via scRNAseq) as compared to protein-level expression (via cell surface marker expression), integration of the transcriptomic and cell surface marker expression data will further refine the signature of engraftable HSCs. Both layers of information at single cell resolution will allow for the identification of novel markers or unique combinations of markers that are directly correlated with engraftment potential. Conclusion: By isolating the GPI-80+ population within the CD34+ fraction in human FL, we have achieved unprecedented resolution of the signature of engraftable HSCs as confirmed by transplantation experiments. The in-depth characterization of this compartment as well as the surrounding CD34+ and CD34- cells within the FL is expected to yield valuable insights with respect to several biological questions. This data can be directly harnessed in improving the purification and expansion of engraftable HSCs as well as in guiding the in vitro generation of HSCs from pluripotent stem cells. Disclosures No relevant conflicts of interest to declare.

Description: Intro: The complex and tightly regulated process of human hematopoietic development culminates in the production of hematopoietic stem cells (HSCs), which subsequently acquire functional competence and undergo expansion in the fetal liver (FL). The establishment of a high-resolution molecular signature of FL HSCs provides insights into HSC biology with potential utility in the purification and expansion of engraftable HSCs ex vivo and the generation of HSCs from pluripotent cell sources. To profile HSCs at this developmental stage, we performed CITE-Seq, a technique that combines single cell RNA sequencing (scRNAseq) and cell surface marker interrogation using oligo-tagged antibodies to simultaneously map transcriptional and protein-level expression in individual cells. To connect expression profiles with functional engraftment, we have coupled this with transplantation assays in immunocompromised mice. Methods: In these studies, three populations of human FL cells were used: CD34- cells, CD34+ cells and CD34+ cells furtherenrichedby expression of GPI-80, a marker tightly linked to engraftment potential, to explicitly identify HSCs capable of long-term engraftment. These populations were stained with a panel of oligo-tagged antibodies, processed via the 10X Genomics platform, and sequenced (26,407 total cells). Results: Transplantation experiments using the same sorted fractions that were assayed by CITE-seq revealed superior engraftment potential of the GPI-80+ fraction, and thus enrichment for bona fide HSCs at the functional level. This functional signature coincided with enrichment for known HSC markers such as ITGA6 (CD49f), PROCR (EPCR), CD164, MLLT3, HLF, CLEC9A and HMGA2 at the transcriptional level. As such, by profiling 〉7000 GPI-80+ cells, we have achieved unprecedented resolution of the engraftable HSC compartment within the FL. Combined analysis of all captured FL fractions accurately recapitulated the hematopoietic landscape of the FL at this developmental stage, representing the expected hematopoietic lineages and cell types. To gain further insight into FL HSCs, we next focused on the CD34+ HSC/progenitor compartment where we tracked cluster dynamics upon functional HSC enrichment between the CD34+ bulk and GPI-80+ sample. We noted a prominent (4-fold) increase in a cluster marked by enrichment for genes including RGCC, LMNA, VIM, ID1 and ID3 as well as components of the AHR pathway, suggesting that this expression profile strongly correlates with engraftment potential. Notably, LMNA is expressed in postnatal HSCs and its expression has been shown to decrease upon aging (Grigoryan et al., 2018). In line with this, we also found that LMNA is more highly expressed in our prenatal FL HSPCs compared to postnatal HSPCs. This data suggests a potential role for LMNA in endowing FL HSCs with their superior engraftment potential compared to postnatal HSCs. To complement our transcriptomic characterization of engraftment potential, we also collected cell surface marker expression data based on sequencing of a series of antibody-derived tags (ADTs). This additional layer of information uncovered expression patterns that weren't readily apparent based on mRNA expression data and inspired us to use this ADT information to gate out populations of interest via in silico sorting. This enabled us to compare the transcriptional profiles associated with well-described HSC enrichment signatures to assess whether they represent equivalent cell populations. We compared CD34+CD90+CD49f+ (~Notta et al., 2011) vs CD34+CD133+GPI-80+ (~Sumide et al., 2018) vs CD34+EPCR+ (~Subramaniam et al., 2019) in silico sorted cells and found a strong overlap in enriched genes, suggesting that the transcriptomic signature corresponding to engraftment potential identified in this work is not exclusive to GPI-80+ sorted cells but represents engraftable HSCs beyond this enrichment strategy. Conclusion: We have achieved unprecedented resolution of the engraftable HSC compartment in the human FL. Combining transcriptional profiling of the engraftment potential of FL HSCs with cell surface level expression data provides an in-depth characterization of this unique and developmentally relevant HSC source. This data will be valuable in optimizing the purification and expansion of engraftable HSCs ex vivo as well as in guiding the in vitro generation of HSCs from pluripotent cell sources. Disclosures No relevant conflicts of interest to declare.

Description: It has recently become clear that in vitro hematopoietic differentiation protocols using pluripotent stem cells mainly capture the primitive wave of hematopoietic development with limited induction of the definitive wave that gives rise to cells with adult-type characteristics such as the ability to express adult β-globin. Recent efforts from our group in optimizing our original differentiation protocol to derive more definitively patterned erythroid cells from iPSCs have resulted in a significant increase in β-globin transcripts as well as robust β-globin protein expression at the most mature stage of differentiation. To better quantify our progress in augmenting β-globin expression and to track globin ontogeny in real-time, we created a β-globin reporter iPSC line that allows for the mapping of β-globin expression throughout erythroid development. To create this tool, TALEN were used to target the β-globin locus in iPSCs where a promoterless GFP cassette was fused in frame to the first codon of the β-globin gene allowing for visualization of β-globin expression at single cell resolution via GFP expression. Interestingly, using our optimized protocol, only about 1% of cells exceeded the GFP detection threshold at the most mature stage of differentiation, suggesting that the several log-fold increase in β-globin transcripts seen at the population level as the cells progress through erythroid differentiation could be the result of high levels of β-globin transcription in just a small fraction of cells. Single cell RNA sequencing of GFP- and GFP+ sorted fractions showed significantly greater levels of β-globin transcripts in the GFP+ fraction in line with this hypothesis, thereby validating our reporter line as a tool to visualize and enrich for β-globin expressing cells. Despite lacking β-globin protein expression, the GFP- cells were not completely devoid of β-globin transcripts. As recent studies suggest that translation is dynamically controlled in maturing red blood cells, our results might indicate that posttranscriptional mechanisms could impact the translation of these globin transcripts. In fact, mining of the genes differentially expressed between both populations revealed several transcripts enriched in the GFP+ fraction that code for proteins involved in the dynamic translational control of transcripts essential for maturing red blood cells. These findings suggest advanced maturation of the GFP+ fraction, as well as a role for posttranscriptional mechanisms in the regulation of β-globin protein expression during erythroid development from iPSCs. Establishment of the developmental time frame of iPSC-derived erythroid cells is challenging as in vitro differentiation cultures lack the spatiotemporal separation of the different hematopoietic waves present in the embryo. Due to the possibility of multiple hematopoietic programs co-existing in one well, bulk expression analyses should be interpreted with caution when used to ascribe primitive or definitive characteristics to cell populations. Considering that the complexity of globin regulation in development might currently be underappreciated due to these factors, we also used single cell RNA sequencing to dissect the globin expression profiles of individual cells. Looking at the co-expression of different globins in individual cells, we found that the majority of the cells express a combination of embryonic (ε), fetal (γ) and adult (β) globins, indicative of a definitive yolk sac identity. Moreover, as β-globin transcripts increased, we observed a decrease in ε-globin transcripts, indicating that primitive/embryonic characteristics are gradually lost as cells gain more definitive/adult features. Further interrogation of the 'β-globin expression signature' that we distilled from the single cell RNA sequencing data will be instructive for future strategies aimed at increasing β-globin protein levels in iPSC-derived erythroid cells. Progress in this area will improve the resolution with which we can study hemoglobinopathies such as β-thalassemia and sickle cell disease and such strategies can then immediately be tested using the β-globin reporter iPSC line as a screening platform. Disclosures No relevant conflicts of interest to declare.