A roadmap from research to clinical testing of mesenchymal stromal cell exosomes in the treatment of psoriasis.

The most clinically trialed cells, mesenchymal stromal cells (MSCs), are now known to mainly exert their therapeutic activity through paracrine secretions, which include exosomes. To mitigate potential regulatory concerns on the scalability and reproducibility in the preparations of MSC exosomes, MSC exosomes were produced using a highly characterized MYC-immortalized monoclonal cell line. These cells do not form tumors in athymic nude mice or exhibit anchorage-independent growth, and their exosomes do not carry MYC protein or promote tumor growth. Unlike intra-peritoneal injections, topical applications of MSC exosomes in a mouse model of IMQ-induced psoriasis alleviate interleukin (IL)-17, IL-23 and terminal complement complex, C5b9 in psoriatic skin. When applied on human skin explants, fluorescence from covalently labeled fluorescent MSC exosomes permeated and persisted in the stratum corneum for about 24 hours with negligible exit out of the stratum corneum into the underlying epidermis. As psoriatic stratum corneums are uniquely characterized by activated complements and Munro microabscesses, we postulated that topically applied exosomes permeate the psoriatic stratum corneum to inhibit C5b9 complement complex through CD59, and this inhibition attenuated neutrophil secretion of IL-17. Consistent with this, we demonstrated that assembly of C5b9 on purified human neutrophils induced IL-17 secretion and this induction was abrogated by MSC exosomes, which was in turn abrogated by a neutralizing anti-CD 59 antibody. We thus established the mechanism of action for the alleviation of psoriatic IL-17 by topically applied exosomes.

Practical considerations in transforming MSC therapy for neurological diseases from cell to EV.

Cell-based therapy using Mesenchymal Stromal Cell (MSC) has generally been efficacious in treating a myriad of diseases in animal models and clinical trials. The rationale for MSC therapy was predicated on the potential of MSC to differentiate and form new replacement cells in the diseased tissue. However, pre-clinical animal and clinical data were more consistent with a secretion- and not a differentiation-based rationale. Analysis of MSC secretion led to the identification of small extracellular vesicles (sEVs) as therapeutically active, secretory agents. MSC-sEVs are defined as bi-lipid membrane vesicles of 50-200 nm in diameter that are secreted by MSCs. They reportedly exert similar therapeutic efficacy as MSCs in many diseases including neurological diseases. MSC-sEVs being small and non-living are intrinsically safer than living MSCs. Manufacturing of MSC-sEVs may also be less complex. Nevertheless, realising the therapeutic potential of MSC-sEVs will require exacting scientific rigor and robustness, as well as compliance to regulatory oversight. This review summarises the scientific rationale for the transition of MSC therapy from a cell- to an EV-based therapy and discusses critical scientific issues in the development of MSC-sEVs therapy.

Assessment of Tumorigenic Potential in Mesenchymal-Stem/Stromal-Cell-Derived Small Extracellular Vesicles (MSC-sEV).

Mesenchymal-stem/stromal-cell-derived small extracellular vesicles (MSC-sEV) have been shown to ameliorate many diseases in preclinical studies. However, translating MSC-sEV into clinical use requires the development of scalable manufacturing processes for highly reproducible preparations of safe and potent MSC-sEVs. A major source of variability in MSC-sEV preparations is EV producer cells. To circumvent variability in producer cells, clonal immortalized MSC lines as EV producer lines are increasingly being used for sEV production. The use of sEVs from immortalized producer cells inevitably raises safety concerns regarding the tumorigenicity or tumor promoting potential of the EV products. In this study, cells from E1-MYC line, a MSC cell line immortalized with the MYC gene, were injected subcutaneously into athymic nude mice. At 84 days post-injection, no tumor formation was observed at the injection site, lungs, or lymph nodes. E1-MYC cells pre-and post-sEV production did not exhibit anchorage-independent growth in soft agar. Daily intraperitoneal injections of 1 or 5 µg sEVs from E1-MYC into athymic nude mice with FaDu human head and neck cancer xenografts for 28 days did not promote or inhibit tumor growth relative to the xenograft treated with vehicle control. Therefore, MYC-immortalized MSCs are not tumorigenic and sEVs from these MSCs do not promote tumor growth.

RSPO2 inhibition of RNF43 and ZNRF3 governs limb development independently of LGR4/5/6.

The four R-spondin secreted ligands (RSPO1-RSPO4) act via their cognate LGR4, LGR5 and LGR6 receptors to amplify WNT signalling1-3. Here we report an allelic series of recessive RSPO2 mutations in humans that cause tetra-amelia syndrome, which is characterized by lung aplasia and a total absence of the four limbs. Functional studies revealed impaired binding to the LGR4/5/6 receptors and the RNF43 and ZNRF3 transmembrane ligases, and reduced WNT potentiation, which correlated with allele severity. Unexpectedly, however, the triple and ubiquitous knockout of Lgr4, Lgr5 and Lgr6 in mice did not recapitulate the known Rspo2 or Rspo3 loss-of-function phenotypes. Moreover, endogenous depletion or addition of exogenous RSPO2 or RSPO3 in triple-knockout Lgr4/5/6 cells could still affect WNT responsiveness. Instead, we found that the concurrent deletion of rnf43 and znrf3 in Xenopus embryos was sufficient to trigger the outgrowth of supernumerary limbs. Our results establish that RSPO2, without the LGR4/5/6 receptors, serves as a direct antagonistic ligand to RNF43 and ZNRF3, which together constitute a master switch that governs limb specification. These findings have direct implications for regenerative medicine and WNT-associated cancers.

Loss-of-Function Mutations in LGI4, a Secreted Ligand Involved in Schwann Cell Myelination, Are Responsible for Arthrogryposis Multiplex Congenita.

Arthrogryposis multiplex congenita (AMC) is a developmental condition characterized by multiple joint contractures resulting from reduced or absent fetal movements. Through genetic mapping of disease loci and whole-exome sequencing in four unrelated multiplex families presenting with severe AMC, we identified biallelic loss-of-function mutations in LGI4 (leucine-rich glioma-inactivated 4). LGI4 is a ligand secreted by Schwann cells that regulates peripheral nerve myelination via its cognate receptor ADAM22 expressed by neurons. Immunolabeling experiments and transmission electron microscopy of the sciatic nerve from one of the affected individuals revealed a lack of myelin. Functional tests using affected individual-derived iPSCs showed that these germline mutations caused aberrant splicing of the endogenous LGI4 transcript and in a cell-based assay impaired the secretion of truncated LGI4 protein. This is consistent with previous studies reporting arthrogryposis in Lgi4-deficient mice due to peripheral hypomyelination. This study adds to the recent reports implicating defective axoglial function as a key cause of AMC.

Katanin p80, NuMA and cytoplasmic dynein cooperate to control microtubule dynamics.

Human mutations in KATNB1 (p80) cause severe congenital cortical malformations, which encompass the clinical features of both microcephaly and lissencephaly. Although p80 plays critical roles during brain development, the underlying mechanisms remain predominately unknown. Here, we demonstrate that p80 regulates microtubule (MT) remodeling in combination with NuMA (nuclear mitotic apparatus protein) and cytoplasmic dynein. We show that p80 shuttles between the nucleus and spindle pole in synchrony with the cell cycle. Interestingly, this striking feature is shared with NuMA. Importantly, p80 is essential for aster formation and maintenance in vitro. siRNA-mediated depletion of p80 and/or NuMA induced abnormal mitotic phenotypes in cultured mouse embryonic fibroblasts and aberrant neurogenesis and neuronal migration in the mouse embryonic brain. Importantly, these results were confirmed in p80-mutant harboring patient-derived induced pluripotent stem cells and brain organoids. Taken together, our findings provide valuable insights into the pathogenesis of severe microlissencephaly, in which p80 and NuMA delineate a common pathway for neurogenesis and neuronal migration via MT organization at the centrosome/spindle pole.

Katanin p80 regulates human cortical development by limiting centriole and cilia number.

Katanin is a microtubule-severing complex whose catalytic activities are well characterized, but whose in vivo functions are incompletely understood. Human mutations in KATNB1, which encodes the noncatalytic regulatory p80 subunit of katanin, cause severe microlissencephaly. Loss of Katnb1 in mice confirms essential roles in neurogenesis and cell survival, while loss of zebrafish katnb1 reveals specific roles for katnin p80 in early and late developmental stages. Surprisingly, Katnb1 null mutant mouse embryos display hallmarks of aberrant Sonic hedgehog signaling, including holoprosencephaly. KATNB1-deficient human cells show defective proliferation and spindle structure, while Katnb1 null fibroblasts also demonstrate a remarkable excess of centrioles, with supernumerary cilia but deficient Hedgehog signaling. Our results reveal unexpected functions for KATNB1 in regulating overall centriole, mother centriole, and cilia number, and as an essential gene for normal Hedgehog signaling during neocortical development.

Unexpected X chromosome skewing during culture and reprogramming of human somatic cells can be alleviated by exogenous telomerase.

Somatic tissues in female eutherian mammals are mosaic due to random X inactivation. In contrast to mice, X chromosome reactivation does not occur during the reprogramming of human female somatic cells to induced pluripotent stem cells (iPSCs), although this view is contested. Using balanced populations of female Rett patient and control fibroblasts, we confirm that all cells in iPSC colonies contain an inactive X, and additionally find that all colonies made from the same donor fibroblasts contain the same inactive X chromosome. Notably, this extreme "skewing" toward a particular dominant, active X is also a general feature of primary female fibroblasts during proliferation, and the skewing seen in reprogramming and fibroblast culture can be alleviated by overexpression of telomerase. These results have important implications for in vitro modeling of X-linked diseases and the interpretation of long-term culture studies in cancer and senescence using primary female fibroblast cell lines.