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.

From Vial to Vein: Crucial Gaps in Mesenchymal Stromal Cell Clinical Trial Reporting.

Retrospective analysis of clinical trial outcomes is a vital exercise to facilitate efficient translation of cellular therapies. These analyses are particularly important for mesenchymal stem/stromal cell (MSC) products. The exquisite responsiveness of MSCs, which makes them attractive candidates for immunotherapies, is a double-edged sword; MSC clinical trials result in inconsistent outcomes that may correlate with underlying patient biology or procedural differences at trial sites. Here we review 45 North American MSC clinical trial results published between 2015 and 2021 to assess whether these reports provide sufficient information for retrospective analysis. Trial reports routinely specify the MSC tissue source, autologous or allogeneic origin and administration route. However, most methodological aspects related to cell preparation and handling immediately prior to administration are under-reported. Clinical trial reports inconsistently provide information about cryopreservation media composition, delivery vehicle, post-thaw time and storage until administration, duration of infusion, and pre-administration viability or potency assessments. In addition, there appears to be significant variability in how cell products are formulated, handled or assessed between trials. The apparent gaps in reporting, combined with high process variability, are not sufficient for retrospective analyses that could potentially identify optimal cell preparation and handling protocols that correlate with successful intra- and inter-trial outcomes. The substantial preclinical data demonstrating that cell handling affects MSC potency highlights the need for more comprehensive clinical trial reporting of MSC conditions from expansion through delivery to support development of globally standardized protocols to efficiently advance MSCs as commercial products.

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.

Transcriptome profiles acquired during cell expansion and licensing validate mesenchymal stromal cell lineage genes.

BACKGROUND: Mesenchymal 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. METHODS: Here, 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. RESULTS: Overall, 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. CONCLUSION: These 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.

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.