Intramuscular administration potentiates extended dwell time of mesenchymal stromal cells compared to other routes.

BACKGROUND: Mesenchymal stromal cells (MSCs) offer great potential for diverse clinical applications. However, conventional systemic infusion of MSCs limits their therapeutic benefit, since intravenously (IV) infused cells become entrapped in the lungs where their dwell time is short. METHODS: To explore possible alternatives to IV infusion, we used in vivo optical imaging to track the bio-distribution and survival of 1 million bioluminescent MSCs administered IV, intraperitoneally (IP), subcutaneously (SC) and intramuscularly (IM) in healthy athymic mice. RESULTS: IV-infused MSCs were undetectable within days of administration, whereas MSCs implanted IP or SC were only detected for 3 to 4 weeks. In contrast, MSCs sourced from human umbilical cord matrix or bone marrow survived more than 5 months in situ when administered IM. Long-term survival was optimally achieved using low passage cells delivered IM. However, MSCs could undergo approximately 30 doublings before their dwell time was compromised. Cryo-preserved MSCs administered IM promptly after thaw were predominantly cleared after 3 days, whereas equivalent cells cultured overnight prior to implantation survived more than 3 months. DISCUSSION: The IM route supports prolonged cell survival of both neo-natal and adult-derived MSCs, although short-term MSC survival was comparable between all tested routes up to day 3. IM implantation presents a useful alternative to achieve clinical benefits from prolonged MSC dwell time at a homeostatic implant site and is a minimally invasive delivery route suitable for many applications. However, optimized thaw protocols that restore full biological potential of cryo-preserved MSC therapies prior to implantation must be developed.

Cytokine Activation Reveals Tissue-Imprinted Gene Profiles of Mesenchymal Stromal Cells.

Development of standardized metrics to support manufacturing and regulatory approval of mesenchymal stromal cell (MSC) products is confounded by heterogeneity of MSC populations. Many reports describe fundamental differences between MSCs from various tissues and compare unstimulated and activated counterparts. However, molecular information comparing biological profiles of activated MSCs across different origins and donors is limited. To better understand common and source-specific mechanisms of action, we compared the responses of 3 donor populations each of human umbilical cord (UC) and bone marrow (BM) MSCs to TNF-α, IL-1ß or IFN-γ. Transcriptome profiles were analysed by microarray and select secretome profiles were assessed by multiplex immunoassay. Unstimulated (resting) UC and BM-MSCs differentially expressed (DE) 174 genes. Signatures of TNF-α-stimulated BM and UC-MSCs included 45 and 14 new DE genes, respectively, while all but 7 of the initial 174 DE genes were expressed at comparable levels after licensing. After IL-1ß activation, only 5 of the 174 DE genes remained significantly different, while 6 new DE genes were identified. IFN-γ elicited a robust transcriptome response from both cell types, yet nearly all differences (171/174) between resting populations were attenuated. Nine DE genes predominantly corresponding to immunogenic cell surface proteins emerged as a BM-MSC signature of IFN-γ activation. Changes in protein synthesis of select analytes correlated modestly with transcript levels. The dynamic responses of licensed MSCs documented herein, which attenuated heterogeneity between unstimulated populations, provide new insight into common and source-imprinted responses to cytokine activation and can inform strategic development of meaningful, standardized assays.

Human umbilical cord perivascular cells: A novel source of the organophosphate antidote butyrylcholinesterase.

Human butyrylcholinesterase (BChE) is a well-characterized bioscavenger with significant potential as a prophylactic or post-exposure treatment for organophosphate poisoning. Despite substantial efforts, BChE has proven technically challenging to produce in recombinant systems. Recombinant BChE tends to be insufficiently or incorrectly glycosylated, and consequently exhibits a truncated half-life, compromised activity, or is immunogenic. Thus, expired human plasma remains the only reliable source of the benchmark BChE tetramer, but production is costly and time intensive and presents possible blood-borne disease hazards. Here we report a human BChE production platform that produces functionally active, tetrameric BChE enzyme, without the addition of external factors such as polyproline peptides or chemical or gene modification required by other systems. Human umbilical cord perivascular cells (HUCPVCs) are a rich population of mesenchymal stromal cells (MSCs) derived from Wharton's jelly. We show that HUCPVCs naturally and stably secrete BChE during culture in xeno- and serum-free media, and can be gene-modified to increase BChE output. However, BChE secretion from HUCPVCs is limited by innate feedback mechanisms that can be interrupted by addition of miR 186 oligonucleotide mimics or by competitive inhibition of muscarinic cholinergic signalling receptors by addition of atropine. By contrast, adult bone marrow-derived mesenchymal stromal cells neither secrete measurable levels of BChE naturally, nor after gene modification. Further work is required to fully characterize and disable the intrinsic ceiling of HUCPVC-mediated BChE secretion to achieve commercially relevant enzyme output. However, HUCPVCs present a unique opportunity to produce both native and strategically engineered recombinant BChE enzyme in a human platform with the innate capacity to secrete the benchmark human plasma form.

Accumulating Transcriptome Drift Precedes Cell Aging in Human Umbilical Cord-Derived Mesenchymal Stromal Cells Serially Cultured to Replicative Senescence.

In preclinical studies, mesenchymal stromal cells (MSCs) exhibit robust potential for numerous applications. To capitalize on these benefits, cell manufacturing and delivery protocols have been scaled up to facilitate clinical trials without adequately addressing the impact of these processes on cell utility nor inevitable regulatory requirements for consistency. Growing evidence indicates that culture-aged MSCs, expanded to the limits of replicative exhaustion to generate human doses, are not equivalent to early passage cells, and their use may underpin reportedly underwhelming or inconsistent clinical outcomes. Here, we sought to define the maximum expansion boundaries for human umbilical cord-derived MSCs, cultured in chemically defined xeno- and serum-free media, that yield consistent cell batches comparable to early passage cells. Two male and two female donor populations, recovered from cryostorage at mean population doubling level (mPDL) 10, were serially cultivated until replicative exhaustion (senescence). At each passage, growth kinetics, cell morphology, and transcriptome profiles were analyzed. All MSC populations displayed comparable growth trajectories through passage 9 (P9; mPDL 45) and variably approached senescence after P10 (mPDL 49). Transcription profiles of 14,500 human genes, generated by microarray, revealed a nonlinear evolution of culture-adapted MSCs. Significant expression changes occurred only after P5 (mPDL 27) and accumulated rapidly after P9 (mPDL 45), preceding other cell aging metrics. We report that cryobanked umbilical cord-derived MSCs can be reliably expanded to clinical human doses by P4 (mPDL 23), before significant transcriptome drift, and thus represent a mesenchymal cell source suited for clinical translation of cellular therapies. Stem Cells Translational Medicine 2019;8:945&958.