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.

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.

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.

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.

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.

Engineered Mesenchymal Cells Improve Passive Immune Protection Against Lethal Venezuelan Equine Encephalitis Virus Exposure.

UNLABELLED: : Mesenchymal stromal cells (MSCs) are being exploited as gene delivery vectors for various disease and injury therapies. We provide proof-of-concept that engineered MSCs can provide a useful, effective platform for protection against infectious disease. Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne pathogen affecting humans and equines and can be used in bio-warfare. No licensed vaccine or antiviral agent currently exists to combat VEEV infection in humans. Direct antibody administration (passive immunity) is an effective, but short-lived, method of providing immediate protection against a pathogen. We compared the protective efficacy of human umbilical cord perivascular cells (HUCPVCs; a rich source of MSCs), engineered with a transgene encoding a humanized VEEV-neutralizing antibody (anti-VEEV), to the purified antibody. In athymic mice, the anti-VEEV antibody had a half-life of 3.7 days, limiting protection to 2 or 3 days after administration. In contrast, engineered HUCPVCs generated protective anti-VEEV serum titers for 21-38 days after a single intramuscular injection. At 109 days after transplantation, 10% of the mice still had circulating anti-VEEV antibody. The mice were protected against exposure to a lethal dose of VEEV by an intramuscular pretreatment injection with engineered HUCPVCs 24 hours or 10 days before exposure, demonstrating both rapid and prolonged immune protection. The present study is the first to describe engineered MSCs as gene delivery vehicles for passive immunity and supports their utility as antibody delivery vehicles for improved, single-dose prophylaxis against endemic and intentionally disseminated pathogens. SIGNIFICANCE: Direct injection of monoclonal antibodies (mAbs) is an important strategy to immediately protect the recipient from a pathogen. This strategy is critical during natural outbreaks or after the intentional release of bio-weapons. Vaccines require weeks to become effective, which is not practical for first responders immediately deployed to an infected region. However, mAb recipients often require booster shots to maintain protection, which is expensive and impractical once the first responders have been deployed. The present study has shown, for the first time, that mesenchymal stromal cells are effective gene delivery vehicles that can significantly improve mAb-mediated immune protection in a single, intramuscular dose of engineered cells. Such a cell-based delivery system can provide extended life-saving protection in the event of exposure to biological threats using a more practical, single-dose regimen.

Nemo promotes Notch-mediated lateral inhibition downstream of proneural factors.

During neurogenesis, conserved tissue-specific proneural factors establish a cell's competence to take on neural fate from within a field of unspecified cells. Proneural genes encode basic helix-loop-helix transcription factors that promote the expression of 'core' and subtype-specific target genes. Target genes include both pan-neuronal genes and genes that aid in the process of refinement, known as lateral inhibition. In this process, proneural gene expression is increased in the neural progenitor while simultaneously down-regulated in the surrounding cells, in a Notch signalling-dependent manner. Here, we identify nemo (nmo) as a target of members of both Drosophila Atonal and Achaete-Scute proneural factor families and find that mammalian proneural homologs induce Nemo-like-kinase (Nlk) expression in cell culture. We find that nmo loss of function leads to reduced expression of Notch targets and to perturbations in Notch-mediated lateral inhibition. Furthermore, Notch hyperactivity can compensate for nmo loss in the Drosophila eye. Thus nmo promotes Notch-mediated lateral inhibition downstream of proneural factors during neurogenesis.

Nemo phosphorylates Eyes absent and enhances output from the Eya-Sine oculis transcriptional complex during Drosophila retinal determination.

The retinal determination gene network comprises a collection of transcription factors that respond to multiple signaling inputs to direct Drosophila eye development. Previous genetic studies have shown that nemo (nmo), a gene encoding a proline-directed serine/threonine kinase, can promote retinal specification through interactions with the retinal determination gene network, although the molecular point of cross-talk was not defined. Here, we report that the Nemo kinase positively and directly regulates Eyes absent (Eya). Genetic assays show that Nmo catalytic activity enhances Eya-mediated ectopic eye formation and potentiates induction of the Eya-Sine oculis (So) transcriptional targets dachshund and lozenge. Biochemical analyses demonstrate that Nmo forms a complex with and phosphorylates Eya at two consensus mitogen-activated protein kinase (MAPK) phosphorylation sites. These same sites appear crucial for Nmo-mediated activation of Eya function in vivo. Thus, we propose that Nmo phosphorylation of Eya potentiates its transactivation function to enhance transcription of Eya-So target genes during eye specification and development.

Nemo phosphorylates Even-skipped and promotes Eve-mediated repression of odd-skipped in even parasegments during Drosophila embryogenesis.

Drosophila nemo (nmo) and other Nemo-like kinase family members (Nlks) are well-established key regulators of numerous conserved signaling pathways, such as Wg and BMP. nmo mutants display pleiotropic defects at different developmental stages, including the embryo. In this study we describe a detailed characterization of embryonic cuticle patterning defects associated with maternal loss of nmo. nmo mutant embryos consistently show segmentation defects, most frequently fusions of pairs of denticle belts in alternating segments. These phenotypes are reminiscent of those associated with defects in pair-rule patterning. Genetic interaction studies demonstrate that Nmo promotes Even-skipped (Eve) activity and is required to promote the expression of the Eve target, engrailed (en), in even numbered parasegments. We find that Nmo regulates a subset of Eve activities by stimulating Eve-mediated suppression of the odd-skipped (odd) repressor. Furthermore, we isolate Nmo in a protein complex with Eve and show that Nmo phosphorylates Eve in in vitro kinase assays. These studies reveal a novel role for the Nmo kinase in embryonic pattern formation through its regulation of the homeodomain-containing transcription factor Eve.

Drosophila nemo promotes eye specification directed by the retinal determination gene network.

Drosophila nemo (nmo) is the founding member of the Nemo-like kinase (Nlk) family of serine-threonine kinases. Previous work has characterized nmo's role in planar cell polarity during ommatidial patterning. Here we examine an earlier role for nmo in eye formation through interactions with the retinal determination gene network (RDGN). nmo is dynamically expressed in second and third instar eye imaginal discs, suggesting additional roles in patterning of the eyes, ocelli, and antennae. We utilized genetic approaches to investigate Nmo's role in determining eye fate. nmo genetically interacts with the retinal determination factors Eyeless (Ey), Eyes Absent (Eya), and Dachshund (Dac). Loss of nmo rescues ey and eya mutant phenotypes, and heterozygosity for eya modifies the nmo eye phenotype. Reducing nmo also rescues small-eye defects induced by misexpression of ey and eya in early eye development. nmo can potentiate RDGN-mediated eye formation in ectopic eye induction assays. Moreover, elevated Nmo alone can respecify presumptive head cells to an eye fate by inducing ectopic expression of dac and eya. Together, our genetic analyses reveal that nmo promotes normal and ectopic eye development directed by the RDGN.