Abstract
BackgroundMesenchymal stromal cells (MSCs) are rapidly advancing as commercial therapeutics. However, there are still no adequate tools to validate the identity of MSCs and support standardization of MSC-based products. Currently accepted metrics include cell surface marker profiling and tri-lineage differentiation assays, neither of which is definitive. Transcript profiling represents a cost- and time-effective approach amenable to MSC manufacturing processes. Two independent labs recently reported non-overlapping MSC-specific transcriptomic signatures of 489 and 16 genes.MethodsHere, we interrogated our repository of transcriptome data to determine whether routine culture manipulations including cell expansion and immune activation affect expression of the reported MSC lineage genes. These data sets comprise 4 donor populations of human umbilical cord (UC) MSCs serially cultured from cryopreservation thaw through pre-senescence, and 3 donor populations each of naïve UC and bone marrow (BM) MSCs and licensed by 3 different cytokines.ResultsOverall, 437 of 456 proposed signature genes assessed in these data sets were reliably expressed, representing an enduring lineage profile in 96% agreement with the previous studies. Serial passaging resulted in the downregulation of 3 signature genes, and one was silenced. Cytokine stimulation downregulated expression of 16 signature genes, and 3 were uniformly silenced in one or the other MSC type. Fifteen additional genes were unreliably detected, independent of culture manipulation.ConclusionThese results validate and refine the proposed transcriptomic tools for reliable identification of MSCs after isolation through cell expansion and after inflammatory activation. We propose a 24-gene signature to support standardized and accessible MSC characterization.
Highlights
Mesenchymal stromal cells (MSCs) are rapidly advancing as commercial therapeutics
Using combinatorial analysis of 285 samples from public data and in-house microarray and RNA-Seq data, Roson-Burgo et al derived an MSC lineage signature of 489 genes based primarily on genes upregulated in the bone marrow (BM), adipose, and placental MSCs compared with hematopoietic stem and progenitor cells [4]
Cells were maintained in TheraPEAKTM MSC growth medium chemically defined (Lonza, MD, USA) and serially propagated until senescence [6] or cultivated in xeno-free human platelet lysate-supplemented media (RoosterBio Inc., MD, USA) and activated by a 24-h co-incubation with TNF-α (50 ng/ml), IFN-γ (50 ng/ml), or IL-1β (80 pg/ml) in protein-free media (RoosterBio Inc.; submitted manuscript)
Summary
Mesenchymal stromal cells (MSCs) are rapidly advancing as commercial therapeutics. there are still no adequate tools to validate the identity of MSCs and support standardization of MSC-based products. Current metrics to establish MSC identity include plastic adherence, cell surface phenotyping, and tri-lineage differentiation [1], which do not clearly distinguish MSCs from other stromal resident cells such as fibroblasts [2] or hepatic stellate cells [3]. The “Rohart MSC test”, an in silico classifier based on 16 MSC signature genes, was created and validated using over 100 transcriptome studies employing 15 different quantification platforms [5]. This test reportedly distinguishes MSCs from non-MSCs with > 95% accuracy [5]
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