Stem Cells Translational Medicine, 2022, 11, 88–96 https://doi.org/10.1093/stcltm/szab007 Original Research Robust In Vitro and In Vivo Immunosuppressive and Anti-inflammatory Properties of Inducible Caspase-9- mediated Apoptotic Mesenchymal Stromal/Stem Cell Paola Alejandra Romecín1,2,∗, Meritxell Vinyoles1, Belén López-Millán1,2,3, Rafael Diaz de la Guardia3, Noemi M. Atucha4, Sergi Querol2,5, Clara Bueno1,2,6, Raquel Benitez7, Downloaded from https://academic.oup.com/stcltm/article/11/1/88/6541839 by guest on 01 February 2023 Elena Gonzalez-Rey7, Mario Delgado7, Pablo Menéndez1,2,6,8,∗ 1 Josep Carreras Leukemia Research Institute, Barcelona, Spain 2 RICORS-TERAV, ISCIII, Madrid, Spain 3 GENYO, Centro Pfizer-Universidad de Granada-Junta de Andalucía de Genómica e Investigación Oncológica, Granada, Spain 4 Departamento de Fisiologia Humana, Facultad de Medicina, Murcia, Spain 5 Banc de Sang i Teixits, Barcelona, Spain 6 CIBERONC, ISCIII, Barcelona, Spain 7 Instituto de Parasitologia y Biomedicina López-Neyra (IPBLN-CSIC), Armilla, Granada, Spain 8 Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain ∗Corresponding author: Pablo Menéndez, Josep Carreras Leukemia Research Institute, School of Medicine, University of Barcelona, Carrer Casanova 143, 4º floor, 08036, Barcelona, Spain. Tel: (+34) 93 5572810; Email: pmenendez@carrerasresearch.org; or, Paola Alejandra Romecin, Josep Carreras Leukemia Research Institute, School of Medicine, University of Barcelona, Carrer Casanova 143, 4º floor, 08036, Barcelona, Spain. Tel: (+34) 93 5572810; Email: promecin@carrerasresearch.org Abstract Mesenchymal stromal stem/cells (MSC) therapies are clinically used in a wide range of disorders based on their robust HLA-independent immunosuppressive and anti-inflammatory properties. However, the mechanisms underlying MSC therapeutic activity remain elusive as dem- onstrated by the unpredictable therapeutic efficacy of MSC infusions reported in multiple clinical trials. A seminal recent study showed that infused MSCs are actively induced to undergo apoptosis by recipient cytotoxic T cells, a mechanism that triggers in vivo recipient-induced immunomodulation by such apoptotic MSCs, and the need for such recipient cytotoxic cell activity could be replaced by the administration of ex vivo-generated apoptotic MSCs. Moreover, the use of MSC-derived extracellular vesicles (MSC-EVs) is being actively explored as a cell- free therapeutic alternative over the parental MSCs. We hypothesized that the introduction of a “suicide gene” switch into MSCs may offer on-demand in vivo apoptosis of transplanted MSCs. Here, we prompted to investigate the utility of the iCasp9/AP1903 suicide gene system in inducing apoptosis of MSCs. iCasp9/AP1903-induced apoptotic MSCs (MSCiCasp9+) were tested in vitro and in in vivo models of acute colitis. Our data show a very similar and robust immunosuppressive and anti-inflammatory properties of both “parental” alive MSCGFP+ cells and apoptotic MSCiCasp9+ cells in vitro and in vivo regardless of whether apoptosis was induced in vivo or in vitro before administering MSCiCasp9+ lysates. This development of an efficient iCasp9 switch may potentiate the safety of MSC-based therapies in the case of an adverse event and, will also cir- cumvent current logistic technical limitations and biological uncertainties associated to MSC-EVs. Key words: BM-MSC; WJ-MSC; iCasp9 switch; immunosuppression; anti-inflammatory; colitis in vivo model. Graphical Abstract Received: 23 July 2021; Accepted: 13 September 2021. © The Author(s) 2022. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Stem Cells Translational Medicine, 2022, Vol. 11, No. 1 89 Introduction without off-target unwanted effects.15,16 The iCasp-9 system comprises unmodified human components, and as shown in Mesenchymal stromal stem/cells (MSC) are multipotent meso- preclinical or clinical studies is unlikely to be immunogenic. dermal progenitor cells that can differentiate into many lin- Here, we prompted to investigate the utility of the iCasp9/ eages of mesenchymal tissues.1,2 They can be isolated from a AP1903 suicide gene system in inducing apoptosis of MSCs. variety of tissues including bone marrow (BM), Wharton Jelly iCasp9/AP1903-induced apoptotic MSCs (MSCiCasp9+) were (WJ), cord blood, amniotic fluid and amniotic membrane, tested in vitro and in in vivo models of acute colitis, and skeletal muscle, adipose tissue, dental pulp, and placenta they show a robust immunosuppressive and anti-inflamma- among others.2,3 Beyond their multi-lineage differentiation tory properties in vitro and in vivo regardless of whether capacity, MSCs lack immunogenicity, and display robust im- apoptosis was induced in vivo or in vitro before adminis- munosuppressive and anti-inflammatory properties, a set of tering MSCiCasp9+ lysates. We report the successful develop- features that have prompted their wide use in cell therapies ment of an efficient iCasp9 switch for improved MSC-based for a range of autoimmune and inflammatory diseases, graft therapies. failure, and graft-versus-host-disease (GvHD) in the context of hematopoietic stem cell transplantation, osteoarticular Downloaded from https://academic.oup.com/stcltm/article/11/1/88/6541839 by guest on 01 February 2023 disorders, heart and pulmonary fibrotic disorders and even SARS-Cov-2.4,5 Although the therapeutic efficacy of MSCs Material and Methods has been demonstrated in preclinical disease models and in Human Primary Cells multiple human phase I/II clinical trials, progress beyond MSCs were sourced from both human BM aspirates from phase III trials has not been achieved.6 pediatric healthy donors obtained from the Catalan Blood The mechanisms underlying MSC therapeutic ac- and Tissue Bank (BST) and from cord blood-derived WJ upon tivity remain elusive.6,7 Our lack of understanding of MSC Institutional Review Board approval (HCB/2014/0687). Both immunobiology underlies the controversial results reported in BM-MSCs and WJ-MSCs were generated and maintained multiple clinical trials showing unpredictable therapeutic effi- using well-established procedures, and characterized mor- cacy of MSC infusions even in homogenously treated patients phologically, phenotypically, and functionally following the suffering from the same disease.6,8,9 Regardless of the MSC- guidelines proposed by the International Society for Cellular specific phenotype and tissue of origin, the cell dose and ad- Therapy.17-19 MSCs were cultured in complete Advanced ministration schedule, the cell delivery route, and the selected DMEM medium (advanced DMEM supplemented with 10% cell product (ie, use of autologous vs allogeneic, freshly cul- heat-inactivated FBS, L-glutamine, and penicillin/strepto- tured vs frozen and thawed MSCs, etc.), a major unresolved mycin (P/S). Bona fide WJ-MSCs and BM-MSCs were used at challenge in the field is posed by the fact that MSCs do not early passages 1-6. seem required to engraft to be efficacious, as indicated by the Peripheral blood mononuclear cells (PBMCs) were isolated fact MSCs are undetectable after in vivo administration.10 from buffy coats of healthy volunteers by Ficoll-Hypaque gra- In fact, an increasing evidence in recent years indicates that dient centrifugation (GE Healthcare, Chicago, IL) obtained extracellular vesicles (EVs) released by MSCs (MSC-EVs) from the BST upon Institutional Review Board approval might exert similar immunosuppressive effects as the MSC (HCB/2014/0687). PBMCs were maintained in complete themselves.11,12 Such potential administration of MSC-EVs as RPMI medium (RPMI-1640 supplemented with 10% heat- a cell-free therapeutic approach may offer several therapeutic inactivated FBS and P/S (all from Gibco/Invitrogen, Waltham, and safety advantages over the parental MSCs.13 MA). A recent seminal study by Dazzi’s Lab based on preclin- ical murine models of GvHD as well as clinical data from Vector Construction and Lentiviral Transduction of MSC-infused GvHD patients demonstrated that the infused MSCs MSCs are actively induced to undergo apoptosis by recipient The AP1903 binding domain fused to iCasp9 (kindly pro- cytotoxic T cells, a mechanism that triggers in vivo recipient- vided by Prof Gianpietro Dotti, University of North Carolina, induced immunomodulation by such apoptotic MSCs.4,14 In NC) before a 2A ribosomal skip sequence followed by a GFP fact, this study also showed that the need for recipient cyto- reporter was subcloned downstream the EF1a promoter in toxic cell activity could be replaced by the administration of our clinically validated pCCL second-generation lentiviral apoptotic MSC generated ex vivo,14 further supporting the backbone20,21 (Fig. 1A). Similarly, the dTomato (dTo)-P2A- use of MSC-EVs as a cell-free therapeutic alternative over the Luciferase (Luc) sequence was also subcloned downstream parental MSCs. the UbC promoter in the pCCL vector (Supplementary Fig. The introduction of a “suicide gene” switch into MSCs S1A). iCasp9/GFP- or dTo/Luc-expressing lentiviral particles may offer on-demand in vivo apoptosis of transplanted pseudotyped with VSV-G were generated by transfection of MSCs. This would potentiate the safety of the infused MSCs HEK 293T cells with pCCL, VSV-G, and psPAX2 vectors in the case of an adverse event but, will also circumvent cur- using standard polyethylenimine (Polysciences, Warrington, rent logistic technical limitations and biological uncertainties PA). Supernatants were collected 48 hours after transfec- associated to MSC-EVs.13 The fusion “inducible Caspase-9” tion and concentrated by ultracentrifugation, as previously suicide gene (iCasp9) was recently engineered by replacing the described.22 Caspase recruitment domain of pro-apoptotic caspase-9 with Semi-confluent WJ-MSCs and BM-MSCs were transduced a mutated dimerizer drug-binding domain from the human in 6-well plates with iCasp9/GFP-expressing lentiviral par- FK506-binding protein. The iCasp9 displays high affinity ticles (or GFP alone as a control) at a multiplicity-of-infection and high specificity for small molecular chemical inducers of 5 in the presence of polybrene (8-10 μg/mL).23,24 Viral of dimerization (CID) such as clinical-grade AP1903, which supernatants were washed out 24 hours later, and transduced effectively induces apoptosis via activation of caspase-9 cells were allowed to recover and expand for 7-15 days. 90 Stem Cells Translational Medicine, 2022, Vol. 11, No. 1 Downloaded from https://academic.oup.com/stcltm/article/11/1/88/6541839 by guest on 01 February 2023 Figure 1. Rapid AP1903-mediated apoptosis of iCasp9-expressing WJ- and BM-MSCs. (A) Schematic of iCasp9/GFP-expressing lentiviral vector. A GFP- expressing vector was used as mock. The iCasp9 cDNA is preceded by an AP1903 binding motif for iCasp9 activation. (B,C) Cell viability measured by GFP expression assessed by fluorescence microscopy and flow cytometry in GFP-expressing (top panels) and iCasp9/GFP-expressing (bottom panels) WJ-MSCs (left) and BM-MSC (right) 16 hours and 24 hours after treatment with 20 nM AP1903. Massive cell death (>95%) was exclusively observed in MSCiCasp9+ treated with AP1903 for 24 hours. (D) Quantification of cell death by GFP assessment after 16 hours and Annexin V staining after 24 hours, n = 5 independent experiments. Data shown as mean ± SEM. ∗∗∗P < .001 relative to DMSO. (E) ELISA quantification of PGE2 levels in WJ-MSCiCasp9+ and BM-MSCiCasp9+ supernatants harvested 24 hours after AP1903 treatment (n = 3). Data shown as mean ± SEM. Then, the GFP+ (MSCGFP+) and iCasp9/GFP+ (MSCiCasp9+) In Vitro AP1903 Apoptotic Assay in WJ and transduced cells were FACS-purified (98% purity) using BM-MSC FACSAria Fusion flow cytometer (BD Biosciences, San Jose, MSCGFP+ and MSCiCasp9+ cells were plated in 6-well plates at CA). The WJ-MSCiCasp9+ cells were further transduced with 0.5 × 106/well, and 48 hours later were treated with the CID Luc/dTo-expressing lentiviral particles, and the resulting AP1903 (20 nM, Bellicum Pharmaceuticals, Houston, TX) WJ-MSCiCasp9+Luc+ were FACS-sorted at purity >94% and es- and/or DMSO as the vehicle. Cells were analyzed by fluores- tablished as described above.23,24 cence microscopy and by flow cytometry 24 hours later, and Stem Cells Translational Medicine, 2022, Vol. 11, No. 1 91 the cell viability was assessed by Annexin V/7AAD apoptosis in stool consistency; 2 = moderate decrease in stool consist- detection kit (BD Biosciences). Cell supernatants were har- ency; 3 = moderate decrease in stool consistency and presence vested in triplicate and analyzed by ELISA assay for quantifi- of blood in stool; and 4 = severe watery diarrhea and mod- cation of prostaglandin E2 (PGE2).24 erate/severe bleeding in the stool.25,28,29 At days 4 and 9 after TNBS injection, sera were collected by cardiac puncture, and In Vitro Immunosuppression of T-cell Response by colons were dissected from caecum to the anus. The levels of MSCGFP+ and MSCiCasp9+ and Cytokine Production the inflammatory cytokines TNFα and IL6 in the sera were MSCGFP+ and MSCiCasp9+ cells (both WJ and BM) were seeded determined by specific ELISAs as previously described.25,27-29 at 2 × 104 cells/well in flat-bottom 96-well plates containing Colons were macroscopically scored for tissue damage signs complete Advanced DMEM medium for 24 hours. The next (scale 0-8), based on the grade of tissue adhesion, presence of day, 1 × 106 cells PBMCs were labeled with 2.5 μM CFSE ulceration and wall thickness: ulceration (0, normal appear- for 20 minutes at 37°C and then washed 3× with complete ance; 1 = focal hyperemia, no ulcers; 2 = ulceration without RPMI medium. Subsequently, the MSC culture medium was hyperemia or bowel wall thickening; 3 = ulceration with in- removed and CFSE-labeled PBMCs were added to the MSC flammation at one site; 4 = two or more sites of ulceration and Downloaded from https://academic.oup.com/stcltm/article/11/1/88/6541839 by guest on 01 February 2023 culture at MSC:PBMC ratios of 1:5 and 1:10 in complete inflammation; and 5 = major sites of damage extending >1 cm RPMI medium in the absence or presence of PHA-L (1 μg/ along length of colon), adhesions (0 = no adhesions; 1 = minor mL). The day after, AP1903 (20 nM) or DMSO as vehicle adhesions, colon can be easily separated from the other tis- was added, and 96 hours later the non-adherent cells were sues; and 2 = major adhesions) and thickness (maximal bowel collected, washed and stained with 7-AAD. Cell proliferation wall thickness, in mm, measured with a caliper).25,27-29 After was measured using CSFE dye dilution on the 7-AAD- vi- macroscopic examination, various colonic segments were able cells by FACS and the number of proliferating cells was fixed in 10% buffered formalin phosphate, paraffin-sectioned determined by gating on the CFSElow subset as previously (5 µm), stained with Masson’s trichrome and Russell-Movat described.25,26 PBMC:MSC co-cultures supernatants were pentachrome solutions and histopathologically analyzed in harvested and analyzed in triplicate by ELISA assay for quan- an Axio Scope A1 microscope (Carl Zeiss) in a blind fashion tification of the pro-inflammatory cytokines IL-2 and IL17A. by 2 independent researchers (5 cross-sections per animal, at 2-mm intervals along colon segment, at 100× magnification). Induction, Treatment, and Analysis of Inflammatory Intestinal inflammation in colon sections was graded from 0 Bowel Disease to 4 as follows: 0 = no signs of inflammation; 1 = low leuco- The animal experiments reported in this study were approved cyte infiltration (1-2 foci); 2 = moderate leucocyte infiltration by the Animal Care and Ethical Committee of Institute with multiple foci; 3 = high leucocyte infiltration, moderate of Parasitology and Biomedicine “López-Neyra”-Spanish fibrosis, high vascular density, thickening of the colon wall, Council of Scientific Research (IPBLN-CSIC, code number: moderate goblet cell loss and focal loss of crypts; and 4 = MDM.03.10/17.CEEA) and performed in compliance with transmural infiltrations, massive loss of goblet cells, exten- the guidelines from Directive 2010/63/EU of the European sive fibrosis and diffuse loss of crypts.25,27,29 Alternatively, in- Parliament on the protection of animals used for scientific flammatory infiltration was quantified in a blinded fashion purposes. Balb/c mice were purchased from Charles River and by counting the number of infiltrating cells in colon mucosa were housed in the specific pathogen-free animal facility of in 5 independent cross-sections using 8 randomly selected IPBLN-CSIC in a controlled-temperature/humidity environ- nonoverlapping high-powered fields (hpf, at 400× magnifica- ment (22 ± 1°C, 60%-70% relative humidity) in individual tion) per section and expressed as the mean of leucocytes/hpf. cages (10 mice per cage, with wood shaving bedding and nesting material), with a 12-hour light/dark cycle and were Statistical Analysis fed with rodent chow (Global Diet 2018, Harlan) and tap All values are expressed as mean ± SEM of mice/experi- water ad libitum. ment. For statistical analysis, Prism GraphPad Prism v9.0 To induce acute colitis, 2,4,6-trinitrobenzene sulfonic acid (GraphPad Prism Software, San Diego, CA) was used. The dif- (TNBS, 2.5 mg) in 50% ethanol (100 μL) was administered ferences between groups (single comparisons) were analyzed intrarectally in 6-8-week-old female Balb/c mice (125 mg by the nonparametric Mann-Whitney U test (for analysis of of TNBS/kg mouse) under light halothane-induced anes- colon damage, colitis, and microscopic scores) or by the un- thesia.27,28 Control mice received 50% ethanol alone. Animals paired Student’s t test (for analysis of cell viability, PBMC were treated intra-peritoneally (i.p.) with DMEM medium proliferation, cytokine levels, and changes in body weight). (untreated mice) or with WJ-MSCGFP+ or WJ-MSCiCasp9+ (106 One-way analysis of variance with Tukey’s post hoc test was cells/mouse, in a volume of 200 μL of DMEM) 12 hours after used when 3 or more experimental groups were compared. instillation of TNBS. Eight hours later, WJ-MSCiCasp9+-treated Survival curves were analyzed by the Kaplan-Meier log-rank animals were injected i.p. with vehicle (1% DMSO in 100 test. We considered P-values <.05 (2-tailed) as significant. μL of DMEM) or AP1903 (5 mg/kg, in 100 μL of DMEM). No data were excluded from the analysis. The variance was Additionally, in another group of animals, treatment consisted similar between the groups that were statistically compared. of the injection i.p. of cell lysates (in a volume of 200 μL of DMEM) obtained from overnight treatment with AP1903 Results (20 nM) of WJ-MSCiCasp9+ cultures (106 cells). Animals were daily monitored for the appearance of diar- Treatment with the CID AP1903 Induces Rapid rhea, body weight loss, and survival. At different time points, and Efficient Apoptosis of WJ-MSCiCasp9+ and colitis signs were scored based on stool consistency and rectal BM-MSCiCasp9+ bleeding by 2 blind observers using the following criteria Early passage, fully characterized WJ- and BM-MSCs17,19 were (scale 0-4): 0 = normal stool appearance; 1 = slight decrease transduced with iCasp9/GFP-expressing (or GFP alone as 92 Stem Cells Translational Medicine, 2022, Vol. 11, No. 1 control) lentivectors (Fig. 1A). Transduced MSCs were FACS- ulceration, and shortening (Fig. 3E). In contrast, mice treated sorted based on GFP expression at high purity (>95%, Fig. with apoptotic WJ-MSCiCasp9+/Luc+ (infusion of WJ-MSCiCasp9+/ 1B,C). When exponentially growing MSCGFP+ and MSCiCasp9+ Luc+ followed by i.p. treatment with AP1903) were largely pro- were treated with AP1903 (20 nM), a massive (approximately tected against colitis, similar to those mice treated with alive 85%-100%) apoptosis/cell death was observed within 16-24 MSCGFP+ cells, showing a significant recovery of their body hours in both WJ- and BM-MSCiCasp9+ but not in WJ- and weight loss, with a significant increase in survival rate (60% BM-MSCGFP+ (Fig. 1B-D). The levels of PGE2, a major bona vs 92%) that was accompanied by the regaining of a healthy fide immunosuppressive factor produced by MSCs, were very appearance, indistinguishable from ethanol-treated control similar in the supernatants of both alive (DMSO-treated) and mice (Fig. 3B,C). Furthermore, the wasting syndrome and the apoptotic (AP1903-treated) WJ- and BM-MSCiCasp9+ (Fig. 1E), signs of colon inflammation were equally improved in mice confirming a sensitive and specific performance of the iCasp9/ treated with either apoptotic WJ-MSCiCasp9+/Luc+ or MSCGFP+ AP1903 switch in human MSCs, in line with that previously cells (Fig. 3D,E). Colitis and colon macroscopic score data reported for induced pluripotent stem cells,16 T-cells,30 and were directly supported by histological colonic examination MSC-based cancer gene therapies.31,32 which showed that apoptotic WJ-MSCiCasp9+/Luc+ significantly Downloaded from https://academic.oup.com/stcltm/article/11/1/88/6541839 by guest on 01 February 2023 reduced TNBS-induced transmural inflammation, depletion of mucin-producing goblet cells, epithelial ulceration, infiltration Apoptotic WJ- and BM-MSCiCasp9+ Robustly of inflammatory cells in the lamina propria, and focal loss of Immunosuppress T-cell Response In Vitro crypts (Fig. 3F). To further confirm the in vivo anti-inflam- MSC are widely recognized for their immunomodulatory po- matory nature of apoptotic WJ-MSCiCasp9+/Luc+, we measured tential, including the inhibition of allogenic T-cell proliferation at day 4 and day 9 after TNBS instillation the levels of the and the production of pro-inflammatory cytokines.33,34 We pro-inflammatory cytokines TNFα and IL6 in the serum and therefore monitored PHA-L-stimulated T-cell division in the found a pronounced decrease in the levels of both cytokines in absence or presence of AP1903-treated WJ- and BM-derived colitis mice treated with apoptotic WJ-MSCiCasp9+/Luc+ as com- MSCGFP+ and MSCiCasp9+ in vitro (Fig. 2A,B). We found that pared to untreated mice (Fig. 3G). Importantly, no statistically apoptotic (AP1903-treated) WJ- and BM-MSC iCasp9+ strongly differences were observed among mice treated with apoptotic inhibited T-cell proliferation, even at a significantly higher ex- WJ-MSCiCasp9+/Luc+ and those treated with alive WJ-MSCiCasp9+/ tent than alive WJ- and BM-MSCGFP+ (Fig. 2C,D). We next Luc+ (without in vivo treatment with AP1903) or WJ-MSCGFP+ analyzed MSC:T-cell supernatants to test whether apoptotic (Fig. 3). Finally, very similar therapeutic effects were observed MSCiCasp9+ also regulates pro-inflammatory cytokine secretion. in colitis mice i.p treated with WJ-MSCiCasp9+/Luc+ cell lysates The analysis of supernatants showed that the levels of IL-2 obtained from overnight treatment of WJ-MSCiCasp9+/Luc+ cells and IL-17A were massively reduced in the presence of apop- with AP1903 (Fig. 3). Collectively, our results show that apop- totic WJ- and BM-MSCiCasp9+ (Fig. 2E,F). Overall, these results totic WJ-MSCiCasp9+/Luc+ are equally capable of suppressing in- show that regardless of the source of origin, the capacity of flammation in vivo than MSCGFP+ cells, largely protecting mice apoptotic MSCiCasp9+ to immunosuppress T-cell response in against acute colitis in a TNBS-induced disease model. vitro is as robust as that displayed by alive MSCGFP+ cells. Robust In Vivo Anti-inflammatory Properties of Discussion Apoptotic MSCiCasp9+ and MSCiCasp9+ Lysates in a MSC therapies are clinically used in a wide range of dis- Mouse Model of Acute Colitis orders. However, although the therapeutic efficacy of MSCs Having confirmed the immunosuppressive properties of apop- has been shown in preclinical animal studies and in human totic MSCiCasp9+ in vitro, we next assessed their anti-inflam- phase I/II clinical trials, progress beyond phase III trials has matory properties in vivo, by harnessing a well-established not been achieved.6 A major challenge in the clinical imple- preclinical model of acute colitis that shares clinical, histo- mentation of MSC therapies is posed by the lack of under- pathological, and immunological features with Crohn’s standing about the mechanisms underlying MSC therapeutic disease (Fig. 3A).35,36 To facilitate the in vivo tracing, activity as demonstrated by the unpredictable therapeutic ef- WJ-MSCiCasp9+ cells were transduced with dTo/Luc-expressing ficacy of MSC infusions reported in multiple clinical trials. In lentiviral particles, and the GFP+/dTo+ double-positive addition, a major unresolved challenge in the field is posed MSCs (WJ-MSCiCasp9+Luc+) were FACS sorted at high purity by the fact that MSCs do not seem required to engraft to (Supplementary Fig. S1A-C). Inflammatory colitis was induced be efficacious, as shown by undetectable MSCs after in vivo by intracolonic administration of TNBS, and mice were i.p. in- infusion.10 Increasing evidence also suggests the use of MSC- fused with either DMEM medium (untreated mice) or with EVs as a cell-free therapeutic alternative with several advan- 106 WJ-MSCGFP+ or WJ-MSCiCasp9+/Luc+ 12 hours after TNBS tages over alive, parental MSCs. However, many biological instillation. WJ-MSCiCasp9+/Luc+-treated animals were i.p treated and scale-up challenges still need to be outdone. In particular, 8h later with either vehicle or AP1903. In line with the in vitro the efficacy and amenability of MSC-EVs are far from being results, in vivo administration of AP1903 successfully induced (pre)-clinically confirmed.13,37 Moreover, preclinical data sug- rapid ablation/apoptosis of WJ-MSCiCasp9+/Luc+ (Supplementary gests an inferior biological effect of MSC-EVs over alive “par- Fig. S1D,E). As expected, TNBS-treated mice developed se- ental” MSCs likely because MSCs exert their function not vere, an acute illness characterized by 40% mortality over a only through EVs but also by direct cell-to-cell contact and 9-day period (Fig. 3B) accompanied by substantial (approxi- through paracrine factors released by MSCs beyond those ex- mately 20%) and sustained body weight loss (Fig. 3C), bloody changed in EVs.38 diarrhea, rectal prolapsed, and pancolitis with the extensive Previous seminal work from Dazzi’s Lab demonstrated wasting syndrome (Fig. 3D). Macroscopic examination of that apoptotic MSCs induce in vivo recipient-mediated colons revealed profound signs of inflammation, hyperemia, immunomodulation and that among MSC-treated patients Stem Cells Translational Medicine, 2022, Vol. 11, No. 1 93 Downloaded from https://academic.oup.com/stcltm/article/11/1/88/6541839 by guest on 01 February 2023 Figure 2. iCasp9/AP1903-induced apoptotic WJ- and BM-MSC robustly immunosuppress T-cell response. (A) Scheme of in vitro T-cell immunomodulation assays with AP1903-treated MSCiCasp9+ and MSCGFP+. (B) Boolean gating flow cytometry strategy to identify proliferating T-cells. (C) Percentage of proliferating T cells measured as % of CFSE+ T cells 6 days after PHA-L stimulation in the absence or presence of AP1903-treated WJ-MSCiCasp9+ and WJ-MSCGFP+. 1:5 and 1:10 MSC:PBMC ratios were used. (D) Identical to panel B but for BM-MSCiCasp9+ and BM-MSCGFP+. (E,F) ELISA quantification of the pro-inflammatory cytokines IL-2 and IL-17A in WJ-MSC:PBMCs (E) and BM-MSC:PBMCs (F) supernatants after 6 days at 1:10 MSC:PBMC ratio. Data show as mean ± SEM. One-way ANOVA test with Turkey’s post hoc test. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001. only those with high cytotoxicity activity against infused replaced by the administration of ex vivo-generated apoptotic MSCs responded well to MSC therapy, thus representing MSCs.4 In the present study, we have developed an iCasp9- a paradigm shift in MSC-based therapies.4 Furthermore, based safety switch for safer MSC-based therapies. iCasp9/ the need for such recipient cytotoxic cell activity could be AP1903-induced apoptotic MSCs (MSCiCasp9+) were tested in 94 Stem Cells Translational Medicine, 2022, Vol. 11, No. 1 Downloaded from https://academic.oup.com/stcltm/article/11/1/88/6541839 by guest on 01 February 2023 Figure 3. In vivo anti-inflammatory properties of iCasp9/AP1903-induced apoptotic MSCs and MSCiCasp9+ lysates in a mouse model of acute colitis. (A) Scheme of the experimental design. Acute colitis was induced in 7-8-week-old mice BalB/c by intracolonic administration of TNBS or ethanol (control). After 24 hours, mice (n = 10-14 per group) were i.p. injected with medium or MSCGFP+ or MSCiCasp9+ (106 cells/mouse). Eight hours after i.p. infusion of MSCiCasp9+ cells, mice were i.p. injected with either vehicle or AP1903 (5 mg/kg). Body weight and survival were followed up for 9 days. WJ-MSCiCasp9+ cells (106) were also treated in vitro with 20 nM AP1903 for 24 hours, and then, the WJ-MSCiCasp9+ lysate was i.p. infused in TNBS-treated mice. (B) Kaplan-Meyer curves showing a significantly improved survival in mice treated with MSCGFP+ or MSCiCasp9+ +/-AP1903, but not with MSCiCasp9+ lysates, compared to TNBS alone group. (C) Daily change in body weight loss relative to day 0 (induction of colon damage). (D) Colitis score determined at days 3, 6, and 9. (E) Macroscopic damage score of the intestine evaluated at days 4 and 9 for the indicated treatments. (F) Left, histopathological scores of colons and number of infiltrating leucocytes in colonic mucosa determined at day 4 for the indicated treatments. Right, representative colon sections stained with Masson’s trichrome and Russell-Movat pentachrome for the indicated treatments. (G) Serum levels of TNFα and IL-6 at days 4 and 9 for the indicated treatments. Data are shown as mean ± SEM. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001 compared to TNBS, 1-way ANOVA test with Turkey’s post hoc test. Stem Cells Translational Medicine, 2022, Vol. 11, No. 1 95 vitro and in in vivo in cutting-edge models of acute colitis. Author Contributions Our data show a robust immunosuppressive and anti-inflam- Designed and performed experiments, analyzed and inter- matory properties of MSCiCasp9+ in vitro and in vivo regard- preted data, prepared figures and wrote the manuscript: less of whether apoptosis was induced in vivo or in vitro P.A.R., M.D. Performed experiments: M.V., B.L.M., R.D.G., before administering MSCiCasp9+ lysates. Apoptotic MSCs are R.B., E.G.R., C.B. Contributed key reagents/cells: S.Q., expected to be engulfed by recipient phagocytes, without af- N.M.A. Conceived the study, designed experiments, inter- fecting the immunosuppressive therapeutic potential of the preted data, wrote the manuscript, and financially supported MSC cargo.4 Although there are more suicide genes, the the study: P.M. iCas9 system has obvious advantages namely: (1) the iCasp9 displays high affinity and high specificity for small molecular CIDs which effectively induce apoptosis via activation of Consent for Publication caspase-9 without off-target unwanted effects,16,31 (2) the All authors have agreed to publish this manuscript. iCasp-9 system comprises unmodified human components, and as shown in preclinical or clinical studies is unlikely to be Downloaded from https://academic.oup.com/stcltm/article/11/1/88/6541839 by guest on 01 February 2023 immunogenic. However, all other suicide gene systems con- Data Availability Statement tain yeast, bacterial or viral expressed proteins, and are likely to be immune targets and noncompatible with long-term per- The data underlying this article are available in the article and sistence in target organs/tissues. The efficiency and specificity in its online supplementary material. of the iCasp9/AP1903 switch here reported in human MSCs is in line with that previously reported for induced pluripo- Supplementary Material tent stem cells,16 T-cells,30 and MSC-based cancer gene ther- apies.31,32 This approach will become of increasing value as Supplementary material is available at Stem Cells Translational clinical applications for MSCs develop further. Medicine online. Conclusion References Despite the similar efficacy of both “parental” alive MSC GFP+ 1. Friedenstein AJ, Chailakhyan RK, Latsinik NV, Panasyuk AF, cells and apoptotic MSCiCasp9+ cells, this development of an ef- Keiliss-Borok IV. 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