Hypoxic Incubation Conditions for Optimized Manufacture of Tenocyte-Based Active Pharmaceutical Ingredients of Homologous Standardized Transplant Products in Tendon Regenerative Medicine

Human fetal progenitor tenocytes (hFPT) produced in defined cell bank systems have recently been characterized and qualified as potential therapeutic cell sources in tendon regenerative medicine. In view of further developing the manufacture processes of such cell-based active pharmaceutical ingredients (API), the effects of hypoxic in vitro culture expansion on key cellular characteristics or process parameters were evaluated. To this end, multiple aspects were comparatively assessed in normoxic incubation (i.e., 5% CO2 and 21% O2, standard conditions) or in hypoxic incubation (i.e., 5% CO2 and 2% O2, optimized conditions). Experimentally investigated parameters and endpoints included cellular proliferation, cellular morphology and size distribution, cell surface marker panels, cell susceptibility toward adipogenic and osteogenic induction, while relative protein expression levels were analyzed by quantitative mass spectrometry. The results outlined conserved critical cellular characteristics (i.e., cell surface marker panels, cellular phenotype under chemical induction) and modified key cellular parameters (i.e., cell size distribution, endpoint cell yields, matrix protein contents) potentially procuring tangible benefits for next-generation cell manufacturing workflows. Specific proteomic analyses further shed some light on the cellular effects of hypoxia, potentially orienting further hFPT processing for cell-based, cell-free API manufacture. Overall, this study indicated that hypoxic incubation impacts specific hFPT key properties while preserving critical quality attributes (i.e., as compared to normoxic incubation), enabling efficient manufacture of tenocyte-based APIs for homologous standardized transplant products.

Atlas of the immune cell repertoire in human atherosclerotic plaques characterized by single cell RNA-sequencing and multi-color flow cytometry

Rationale Atherosclerosis is a chronic inflammatory disease that is driven by the accumulation of pro- and anti-inflammatory leukocytes in the intima of affected arteries. Yet, the cellular composition of human atherosclerotic plaques is only poorly understood. Here, we characterized immune cells to human carotid atherosclerotic plaques by multi-color flow cytometry and scRNAseq. Methods and results First, we compared a set of previously reported digestion protocols to liberate leukocytes from human carotid plaques after surgical thrombendarteriectomy. One digestion cocktail, containing Collagenase IV and DNase I, was superior regarding cell survival and cell surface marker preservation. Second, leukocytes from 56 surgical specimen were characterized by flow cytometry with a set of 16 parameters and cell surface markers capable of identifying principal hematopoietic leukocyte lineages. This protocol allowed to extract and analyze on average 4x103 viable CD45+ leukocytes from a mean of 988 mg plaque tissue. Surprisingly, we found that atherosclerotic plaques were dominated by T cells with 33.7±2.2% CD4+ T-helper cells and 25.6±2.5% CD8+ cytotoxic T cells. CD11b+ myeloid cells, including monocytes and macrophages, represented only 20.2±4.0% of all CD45+ leukocytes. CD19+B cells and CD56+ NK-cells accounted for 3.9±1.2 and 3.3±0.5%, respectively. TCR-g/d+ T cells and neutrophils were undetectable in atherosclerotic plaques. This cellular composition differed significantly from peripheral blood, but was not relevantly changed between different plaque locations, indicating that macrophage-rich necrotic cores mostly contain dead cells. We confirmed the principal composition of human plaques by single-cell RNA-sequencing from six patients. To allow an estimation of cellular heterogeneity independent of classical cell surface marker assignment, we performed an unsupervised cluster detection algorithm by t-distributed stochastic neighbor embedding (tSNE) and found more than 16 leukocyte clusters with unique cell surface marker expression, suggesting an unexpected high diversity of plaque leukocytes. Conclusion We developed an immune cell phenotyping protocol optimized for human carotid plaques. The definition of phenotypes and frequencies in atherosclerotic plaques will allow to build clinical associations between the immune cell composition and clinical outcomes in future. Funding Acknowledgement Type of funding source: None

Transcriptional, Protein-Level and Functional Profiling of Human Fetal Liver-Derived Hematopoietic Stem Cells at Single Cell Resolution

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.