In Vivo-in Vitro Genomic Comparison Reveals TNFα As a Missing Factor That Supports in Vitro Hematopoiesis
Authors: Qiu Rong , Shalaby Carson , Garifallou James , Thom Christopher S. 
Published: December 2024

Background
Inefficient blood cell production in vitro is a major hurdle for blood cell-based cell therapeutics development. In vitro systems can differentiate induced pluripotent stem cells (iPSCs) into hemogenic endothelial cells (HECs), which are direct precursors to hematopoietic stem and progenitor cells (HSPCs) that can form multiple blood lineages. Stromal and vascular endothelial cells are also generated in this culture system, but may not fully recapitulate cell types and niche factors that otherwise support HEC and HSPC formation in vivo.
Objectives
This study aimed to use single cell genomics approaches to identify factors that support in vivo HSPC formation that were absent in a definitive in vitro system. We hypothesized that we could replete missing in vivo factors to augment in vitro HSPC yield.
Design/Methods
We created a multiomics data set from a definitive iPSC hematopoiesis model, which we and others have confirmed to produce functional HSPCs with multilineage potential. We used publicly available single cell sequencing pipelines, including Seurat and CellChat, to analyze single cell data. We used the definitive iPSC models to functionally validate our findings.
Results
To identify factors that support in vivo HSPC yield, we used CellChat to define 34 significant cell-cell interactions pathways among public single cell RNAseq data from human embryonic cells during HEC and HSPC formation (Calvanese et al, Nature 2022, padj&lt;0.05). Among these pathways were TGFβ, VEGF, CXCL, and Wnt signaling from stroma and mesenchymal cells to HECs. These pathways all support HEC formation and function. We also identified inflammatory (TNFα) and metabolic (Visfatin, Osteopontin) signals from circulating hematopoietic cells that interacted with ligand receptors present on HECs.
We hypothesized that some of these signaling modalities might be deficient in vitro, limiting HSPC formation. Single cell RNAseq of a clinically relevant in vitro hematopoietic system revealed that, while the culture system retained many key cell interaction activities, TNFα signaling was absent during nascent HSPC formation. This was due to deficiency in TNFα ligand-expressing cells, as TNF receptors were highly expressed on HECs compared with other cells produced in the in vitro system. Functional deficiency in TNFα signaling was confirmed by adding exogenous recombinant TNFα to cultures during HEC and HSPC formation. Addition of exogenous doubled functional HSPC yield (p&lt;0.05). Increasing doses of TNFα (50 pg/mL during HEC formation) increased formation of HSPCs biased toward myeloid lineage.
Conclusions
Our results reveal systemic and niche signaling factors that support hematopoiesis at a single cell level. Timely TNFα addition can bring in vitro HSPC formation closer to clinical efficiency, provided that minimal dose and duration are used. Further augmenting HEC and HSPC production via targeted genetic or environmental manipulation will enhance in vitro derivation of blood cell-based therapeutics. More broadly, our in vivo-in vitro comparison approach at the single cell level may facilitate organoid development for genetic disease modeling in other systems.