Embryonic Stem Cell‐Derived Mesenchymal Stem Cells (MSCs) Have a Superior Neuroprotective Capacity Over Fetal MSCs in the Hypoxic‐Ischemic Mouse Brain

Abstract Human mesenchymal stem cells (MSCs) have huge potential for regenerative medicine. In particular, the use of pluripotent stem cell‐derived mesenchymal stem cells (PSC‐MSCs) overcomes the hurdle of replicative senescence associated with the in vitro expansion of primary cells and has increased therapeutic benefits in comparison to the use of various adult sources of MSCs in a wide range of animal disease models. On the other hand, fetal MSCs exhibit faster growth kinetics and possess longer telomeres and a wider differentiation potential than adult MSCs. Here, for the first time, we compare the therapeutic potential of PSC‐MSCs (ES‐MSCs from embryonic stem cells) to fetal MSCs (AF‐MSCs from the amniotic fluid), demonstrating that ES‐MSCs have a superior neuroprotective potential over AF‐MSCs in the mouse brain following hypoxia‐ischemia. Further, we demonstrate that nuclear factor (NF)‐κB‐stimulated interleukin (IL)‐13 production contributes to an increased in vitro anti‐inflammatory potential of ES‐MSC‐conditioned medium (CM) over AF‐MSC‐CM, thus suggesting a potential mechanism for this observation. Moreover, we show that induced pluripotent stem cell‐derived MSCs (iMSCs) exhibit many similarities to ES‐MSCs, including enhanced NF‐κB signaling and IL‐13 production in comparison to AF‐MSCs. Future studies should assess whether iMSCs also exhibit similar neuroprotective potential to ES‐MSCs, thus presenting a potential strategy to overcome the ethical issues associated with the use of embryonic stem cells and providing a potential source of cells for autologous use against neonatal hypoxic‐ischemic encephalopathy in humans. Stem Cells Translational Medicine 2018;7:439–449


INTRODUCTION
Mesenchymal stromal/stem cells (MSCs) are multipotent cells found in various tissues throughout development, where they contribute to the repair and regeneration of these tissues. Human fetal MSCs have been isolated from various fetal tissues including first trimester blood, liver, or bone marrow (BM) [1][2][3], or from extraembryonic tissues such as the placenta, umbilical cord or amniotic fluid [4,5]. Due to their more primitive phenotype, fetal MSCs present various advantages over their adult counterparts such as longer telomeres, active telomerase and a greater expansion capacity [6]. MSCs, as defined by their plastic adherence, tri-lineage differentiation potential, co-expression of the cell surface markers CD105, CD90, and CD73 and absence of expression of CD34 and CD45 [7], are currently under assessment in numerous clinical trials worldwide targeting a range of disorders from myocardial infarction to stroke [8]. However, the specific mechanisms by which they mediate tissue regeneration in a range of disease contexts remain largely elusive.
Increasing evidence suggests that the therapeutic potential of MSCs should not be attributed only to their engraftment and differentiation but also to the release of regenerative paracrine/ endocrine factors. These effects of MSCs were first suggested when, after injection, BM-MSCs exerted therapeutic effects on the infarcted heart despite being detected largely in the lungs and liver in various murine heart disease models [9,10]. Furthermore, MSC-conditioned medium (CM) has been shown to exert a therapeutic effect in animal models of cardiomyopathy [11] and MSCderived exosomes have been shown to ameliorate disorders of the brain, kidney, and joints [12][13][14][15]. These studies are paving the way for off-the-shelf cell-free therapies, which would have the benefits of being more well defined and standardized than stem cell treatments.
Human primary MSCs, especially those from aged donors, have a limited proliferation capacity in vitro due to replicative senescence [32][33][34] and higher passage MSCs are more likely to trigger an innate immune attack upon transplantation into humans [35]. In addition, cells derived from aged donors tend to be more pro-inflammatory than those from younger donors [36][37][38], thereby further restricting the clinical potential of expanded human primary cells. To address these issues, various groups have generated pluripotent stem cell-derived MSCs (PSC-MSCs), of which there are two types, embryonic stem cell (ESC)derived MSCs (ES-MSCs) [39][40][41] and induced pluripotent stem cell (iPSC)-derived MSCs (iMSCs). Pluripotent stem cells can be expanded in vitro without undergoing senescence and PSC-MSCs have been shown to have enhanced therapeutic potential compared with other sources of MSCs in animal models of various diseases including multiple sclerosis [42], cardiomyopathy [11], pulmonary arterial hypertension [43], osteoarthritis [14], and limb ischemia [44], potentially due to their "rejuvenated" phenotype [45]. However, some studies report comparable [46][47][48] or even decreased [45] immunosuppressive abilities of PSC-MSCs over other sources of MSCs.
In this study, we compare for the first time the neuroprotective potential of ES-MSCs and fetal amniotic fluid-derived (AF)-MSCs. We show that ES-MSCs have an increased neuroprotective capacity compared with AF-MSCs in a perinatal mouse hypoxia-ischemia model. Further, we demonstrate in vitro that increased NF-jB activation in ES-MSCs leads to elevated interleukin (IL)-13 production, thus providing a potential mechanism for the enhanced neuroprotective capacity of ES-MSCs over AF-MSCs. Finally, we compare these two MSC lines to iMSCs, showing that iMSCs also exhibit increased NF-jB activation, concomitant with elevated IL-13 production and increased antiinflammatory potential in comparison to AF-MSCs. Together, these findings suggest that PSC-MSCs are a promising cell source for use in therapeutic strategies targeting neonatal hypoxic-ischemic encephalopathy (HIE) in humans.

Ethics Statements
The healthy donors who provided the amniotic fluid in this study provided written informed consent in accordance with the Declaration of Helsinki.

Cell Culture
Human AF-MSCs were isolated as previously described [4]. Unless specified, cells were plated at 1 3 10 4 cells per cm 2 in D10 (Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 IU/ml penicillin, and 50 mg/ ml streptomycin [all Sigma-Aldrich, Dorset, UK]) at 378C with 5% CO 2 . Where indicated, 20 ng/ml TNFa (Sigma-Aldrich), 125 ng/ml IL-13P (Santa Cruz Biotechnology, CA, USA) or an equal volume of the appropriate vehicle was added to the cells in D10. The human ESC line H1 (WiCell Research Institute, Madison, USA) was cultured in feeder-free conditioned on Matrigel-coated plates in mTESR (Stem Cell Technologies, Cambridge, UK). iPSC Reprogramming of AF-MSCs 5 3 10 5 AF-MSCs were detached using TrypLE (Life Technologies, Paisley, UK), centrifuged at 1,200 rpm for 5 minutes then electroporated as described previously [49] but without the pCXWB-EBNA1 episomal plasmid. After transfection, cells were plated onto one 0.1% gelatin (Sigma-Aldrich)-coated well of a 6-well plate. Twenty-four hours post-transfection, cells were detached and seeded into one 6 cm 2 dish precoated with Matrigel (Scientific Laboratory Supplies ltd, Nottingham, UK) in Essential 8 medium (Life Technologies). The medium was then replenished every 2 days until around day 20, when iPSC colonies were passaged manually with a needle into a fresh Matrigel-coated well of a 6-well plate in Essential 8 medium. Cells were passaged with 0.5 mM EDTA (Life Technologies) thereafter.

MSC Differentiation of Pluripotent Stem Cells
PSC-MSC differentiation was performed as described in Chen et al. [50] with some modifications. Three iMSC and three ES-MSC lines were generated. Briefly, a confluent T25 of ESCs or iPSCs was treated with 10 mM SB431542 for 10 days before the cells were passaged using Tryple Select (Life Technologies) at a 1:1 ratio into StemMACS MSC expansion medium supplemented with CytoMix according to the manufacturer's instructions (both Miltenyi Biotech, Bisley, UK). Cells were subsequently seeded at 2 3 10 4 cells per cm 2 at the first passage, then 1 3 10 4 cells per cm 2 thereafter and analyzed around passage 6.

AF-MSC Transfection
Cells were electroporated as described above in electroporation buffer containing 2 mg each of either pCMV-IKK2 or the pCMV-FLAG control plasmid (both Addgene, Cambridge, UK).

Embryoid Body Formation
iPSCs were detached using EDTA and resuspended in Essential 8 medium containing 10 mM Y-27632 (Sigma-Aldrich) in bacterial culture dishes (non-TC treated). The medium was replaced on day 2, then changed to D10 containing 20% FBS (D20) on days 4 and 6. On day 7, the embryoid bodies were plated onto 0.1% gelatin (Sigma-Aldrich). The cells were fed every 2 days with D20 until being fixed with 4% PFA on day 14.

Lentiviral Production and Transduction
HEK 293T cells were plated at 80% confluency in one well of a 6well plate. They were then transfected using poly polyethylenimine precomplexed with 2.79 mg of the appropriate lentiviral vector along with 0.96 mg VSVg envelope plasmid and 1.79 mg packaging plasmid (pCMVD8.74) in OptiMEM (Life Technologies) for 3 hours prior to re-feeding. Culture medium was then harvested at 48 hours post-transfection and filtered through a 0.45 mm PVDF filter (Elkay, Basingstoke, UK) on to one well of receiver cells.

NF-jB Anti-Inflammatory Assay
HEK 293T cells were transduced with the pLNT-NF-jB-NLuc (NLuc) and pLNT-SFFV-VLuc (VLuc) lentiviral plasmids as previously described [49]. Reporter cells were then plated in triplicate in 24well plates. Twenty-four hours later, cells were activated with 20 ng/ml TNFa (Sigma-Aldrich). Twenty-four hours after activation, 12 ml CM from 6 wells of cells was concentrated in an Amicon Ultra-15 3K NMWL filter (Millipore, Watford, UK) by centrifugation at 4,000g for 30 minutes. The cells were then counted and the volume of medium added was adjusted according to cell number. The appropriate volume of concentrated medium was added to 1.5 ml D10. 0.5 ml was then added to each well of reporter HEK 293T cells in triplicate. Twenty-four hours after the addition of CM the medium was replaced with fresh D10 and luciferase assays were performed following a further 24 hours.

RT-PCR
Total RNA was isolated and reverse transcribed to cDNA as previously described [49]. Reverse transcription (RT)-PCR was then performed using 1 ml cDNA which was amplified for 30 cycles at 548C using the appropriate primer pair (Table 1). Samples were separated using a 1.5% agarose gel and visualized using a UV transilluminator.

qRT-PCR
Total RNA was isolated and reverse transcribed to cDNA as previously described [49]. Triplicate samples containing the appropriate primers (1 mM, Table 2) were analyzed by quantitative (q)RT-PCR using SYBR Green PCR Master Mix (Life Technologies) according to the manufacturer's instructions.

Flow Cytometry
Cells were detached as described previously and washed in flow buffer (phosphate-buffered saline (PBS) 1 1% BSA). Cells were then centrifuged at 5,000g for 2 minutes before 1 3

Luciferase Assays
The supernatant was collected from triplicate wells of cells at the appropriate time points and 20 ml was transferred to 20 ml assay buffer (25 mM Tris Phosphate [pH7.8] containing 1% BSA and 30% glycerol, all Sigma-Aldrich) in a white-bottomed 96-well plate (Corning, NY, USA) in technical triplicates. VLuc samples were assayed detecting photonic emissions at 460 nm after addition of 5 nM vargulin (Gold Biotechnology, Olivette, USA) and NLuc photonic emissions at 454 nm after addition of 2 mM coelenterazine (Gold Biotechnology) which had been incubated on ice for 45 minutes with 0.1 M KI and 10 mM EDTA (Sigma-Aldrich) using a FLUOstar Optima luminometer (BMG Labtech, Ortenberg, Germany). NLuc values were divided by VLuc values before the average fold change over control 6 standard deviation was plotted graphically.
p values were calculated using analysis of variance (One-way ANOVA) followed by Bonferroni's multiple comparison post hoc test.

Immunofluorescent Cell Staining
Immunocytochemistry was performed as previously described [49]. Briefly, cells were washed with PBS and fixed in situ using 4%

Enzyme-Linked Immunosorbent Assays
Cells were plated at 2 3 10 4 cells per cm 2 in four wells of a 6-well plate. Seventy-two hours after seeding, 8 ml CM was removed and concentrated using an Amicon ultra-4 15ml 3K NMWL filter by centrifugation at 4,000g for 40 minutes. 300 ll of D10 was then added to the concentrated medium and 100 ml of this was added per well of the enzyme-linked immunosorbent assay (ELISA) plate. The mini ABTS ELISAs (Peprotech, London, UK) were performed as per the manufacturer's instructions.

Hypoxic-Ischemic Induction
All animal experiments and care protocols were carried out according to the U.K. Animals (Scientific Procedures) Act 1986 and approved by the Home Office. The ARRIVE guidelines were followed. All experiments involved C57/Bl6 mice obtained from Charles River (Kent, U.K.). The surgical procedures were performed as previously described [51]. Briefly, male and female postnatal day 7 (p7) mice were anesthetized with isofluorane (5% induction followed by 1.5% maintenance) before the left common carotid artery was permanently ligated. The mice were then returned to the dam for 2 hours before being exposed to 8% oxygen/92% nitrogen at 368C for 1 hour. Immediately after hypoxic-ischemic (HI) insult, 1 3 10 5 AF-MSCs or ES-MSCs were then injected in 2 ml PBS through a single contralateral injection taking approximately 60 seconds.

Tissue Sample Preparation
Animals were sacrificed at 48 hours post-injection by intraperitoneal injection of pentobarbitane and perfused as described in Hristova et al. [51] Briefly, following sacrifice, animals were perfused with 30 ml 4% paraformaldehyde (PFA) in PBS. Brains were then isolated, fixed in 4% PFA for 1 hour at 48C and then cryopreserved for 24 hours in phosphate-buffered sucrose solution before being frozen in dry ice, cut on a cryostat into 40 mm sections and stored at 2808C until required for analysis.

Immunohistochemistry and Histological Analysis
Five cryosections from each brain (400 mm apart) were rehydrated in H 2 O and then stained as previously described [51]. Infarct volume measurement was performed on cresyl violet-stained sections. These sections were imaged using a Zeiss Axiplan2 Axiocam HRc camera and imported into Image J, which was then used to determine the area of the following bilateral regions: cortex (CTX), pyriform cortex (PYR), hippocampus (HPP), striatum (STR), thalamus (THAL), and external capsule (EC). The percentage tissue loss in comparison to the control side was then calculated by converting the injured and uninjured areas into mm 2 then converting to volume by multiplying by 400 mm. To determine the levels of microglial activation, brain tissue was stained using an anti-aMb2 integrin antibody (1:5,000, Bio-Rad, Kidlington, UK) and semiquantitative scores were allocated to each brain region (CTX, PYR, HPP, STR, THAL, and EC) by an observer blinded to the treatment of the groups, as previously described [51,52], to describe microglial appearance, that is, 0 (no activation), 1 (focal microglial activation), 2 (mild phagocytic activation affecting <50% of the region), 3 (phagocytic activation affecting >50% of the region), and 4 (total phagocytic activation). To determine levels of astroglial activation, sections were stained using an anti-glial fibrillary acidic protein (GFAP) antibody (1:6,000, Dako, Glostrup, Denmark) before three 320 images per region of both the ipsilateral and contralateral sides were captured. Images were then imported into Image J which was used to obtain the mean and standard deviation for the optical luminosity values (OLVs). The standard deviation was subtracted from the values obtained for the surrounding glass slide. Images were contrast stretched using Adobe Photoshop 6.0.

Statistical Analysis
Statistical analysis was performed using SPSS. One-way analysis of variance (ANOVA)s followed by Bonferroni's multiple comparison post-hoc test or Kruskal-Wallis nonparametric tests were used to obtain p values where appropriate. p < .05 was significant.

Validation of ES-MSC Identity
We modified the protocol published by Chen et al. [50] to differentiate ESCs into ES-MSCs. To validate this protocol, we confirmed that the ES-MSCs had gained a characteristic MSC-like morphology (Fig. 1Ai) along with the expression of the MSC markers CD105, CD73, CD90 (Fig. 1Aii), and CD29 (Supporting Information Fig.  S1A), and lacked the expression of the hematopoietic markers CD45 and CD14 (Supporting Information Fig. S1B, S1C). We also confirmed the loss of pluripotency marker expression (SOX2, OCT4A, and NANOG) in these cells by RT-PCR (Fig. 1B).

ES-MSCs Have Enhanced Neuroprotective Potential in Comparison to AF-MSCs
Various groups [44,45,53] have previously documented the increased proliferative potential of PSC-MSCs in comparison to BM-MSCs but we are the first to compare PSC-MSCs to fetal MSCs. We observed that ES-MSCs proliferate at an increased rate in comparison to amniotic fluid-derived MSCs (Fig. 1C). Numerous groups have demonstrated the improved immunoregulatory potential of PSC-MSCs in comparison to the parental population [11,14,53]. In support of these findings, we observed that the media conditioned by ES-MSCs (ES-MSC-CM) had significantly higher anti-inflammatory potential than that derived from AF-MSCs in an in vitro NF-jB reporter assay, using a lentiviral reporter system previously described by us [49] (Fig. 1Di, 1Dii)). Suppression of NF-jB activation in reporter cells by MSCs or their CM or exosomes has been used by various other groups previously to demonstrate their anti-inflammatory potential [54][55][56]. Importantly, neither concentrated D10 medium alone or medium conditioned by ESCs or iPSCs decreased NF-jB activation reporter cells, thus confirming that the effect is specific to MSC-CM (Supporting Information Fig. S2A).
To determine whether the increased in vitro antiinflammatory potential of PSC-MSC-CM translates to an increased therapeutic potential in vivo, we injected the cells into the contralateral side of the brains of neonatal (postnatal day 7) mice subjected to HI insult. Both AF-MSCs and ES-MSCs demonstrated the ability to reduce forebrain tissue loss across all examined regions, with the most notable decrease observed in the cortex, pyriform cortex, striatum, and external capsule (Fig. 2Ai). In all examined regions except the thalamus, ES-MSCs demonstrated a tendency to reduce tissue loss to a higher degree than AF-MSCs reaching significance in the cortex, pyriform cortex, and overall (Fig. 2Ai). Representative images of the three treated groups (PBS, AF-MSC, and ES-MSC) are shown in Figure 2Aii. Both cell types also reduced HI-induced microglial activation. The reduction achieved by ES-MSC injection reached significance in all examined regions and overall whereas the reduction achieved by AF-MSC treatment only reached significance in the striatum (Fig. 2Bi). Notably, ES-MSCs exhibited a tendency to reduce microglial activation to a higher degree than AF-MSCs in all examined regions, reaching significance in the thalamus and overall (Fig. 2Bi). Representative images of the levels of microglial activation in all three groups are shown in Figure 2Bii. We observed that MSC injection was able to prevent the transition of microglia from a ramified, inactive morphology to the round, phagocytic phenotype observed in the PBS control group (Fig. 2Biii).
A similar effect was observed for astroglial activation assessed through GFAP immunoreactivity. We observed a trend toward reduced levels of GFAP immunoreactivity in response to HI insult in the MSC-treated groups in all examined regions of the forebrain except the striatum, where only ES-MSCs decreased astroglial activation (Fig. 2Ci). Again, ES-MSCs showed a trend toward reducing GFAP immunoreactivity to a higher degree than AF-MSCs, with this difference reaching significance in the striatum (Fig. 2Ci). No significant differences were observed between treatments in the contralateral side (data not shown). We observed reduced overall GFAP immunoreactivity in response to treatment with either type of MSCs over the PBS control (Fig. 2Cii) and substantially reduced and more punctate staining of GFAP1 astroglial processes in the ES-MSC treatment group compared with the other two treatment groups (PBS and AF-MSC) (Fig. 2Ciii).

ES-MSCs Exhibit Higher Levels of NF-jB Activation than AF-MSCs
Since NF-jB activity has previously been linked to the therapeutic potential of MSCs [28][29][30], we then determined whether the increased anti-inflammatory potential of ES-MSCs compared with AF-MSCs was due to increased NF-jB activation leading to increased production of anti-inflammatory cytokines. We performed qRT-PCR for three NF-jB target genes, NF-jB1, IKK2, and RelA, in MSCs under basal conditions and observed that the levels of expression of these transcripts were higher in ES-MSCs compared with AF-MSCs (Fig. 3A). Additionally, Western blotting revealed an increase in IKK2 protein expression in ES-MSCs compared with AF-MSCs (Fig. 3B) and the levels of NF-jB activation following TNFa stimulation of NF-jB reporter MSCs were also higher in these cells, as assessed by a luciferase assay (Fig. 3C).
To show that these observations were not specific to the origin of the MSCs we also demonstrated increased activation of NF-jB in  Fig. S2B). BM-iMSC-CM also showed a tendency toward having increased anti-inflammatory potential compared with BM-MSC-CM (Supporting Information Fig. S2C).

ES-MSCs Secrete Increased Levels of anti-Inflammatory Cytokines Compared with AF-MSCs
We then aimed to identify the anti-inflammatory cytokines, downstream of NF-jB, that contribute to the increased antiinflammatory potential of ES-MSC-CM compared with AF-MSC-CM. A cytokine array identified increases in the production of IL-13, IL-1ra and macrophage inhibitory factor (MIF) by ES-MSCs compared with AF-MSCs (Fig. 3D). Since IL-13 and IL-1ra are NF-jB target genes we investigated the secretion of these cytokines further using ELISAs and confirmed that both IL-13 and IL-1ra were released at significantly higher levels by ES-MSCs than AF-MSCs (Fig. 3Ei, 3Eii).

NF-jB-Induced IL-13 Production Contributes to the Increased anti-Inflammatory Potential of ES-MSCs Compared with AF-MSCs
To determine whether elevated NF-jB activation causes increased secretion of IL-13 and IL-1ra in MSCs, we used either TNFa stimulation or overexpression of IKK2 to activate NF-jB signaling in AF-MSCs. We validated the ability of the IKK2 expression plasmid to increase NF-jB activation in AF-MSCs using a luciferase assay (Fig.  4Ai) and qRT-PCR for NF-jB target gene expression (Fig. 4Aii). We then used an ELISA to determine whether NF-jB activation by either TNFa stimulation or overexpression of IKK2 causes increased IL-13 or IL-1ra production. We found that increasing NF-jB activation by either of these methods led to increased levels of IL-13, but not IL-1ra, production in AF-MSCs (Fig. 4Bi, 4Bii).
Finally, to determine whether NF-jB-induced IL-13 production contributes to the increased anti-inflammatory potential of ES-MSCs compared with AF-MSCs we blocked IL-13 production in ES-MSCs using an inhibitory peptide (IL-13P). Treatment with the IL-13P resulted in the decreased anti-inflammatory potential of PSC-MSC-CM (Fig. 4C) thus indicating that, as predicted, NF-jBstimulated IL-13 production contributes to the increased antiinflammatory potential of ES-MSCs compared with AF-MSCs. A schematic for the proposed link between NF-jB activation and IL-13 production in PSC-MSCs versus other types of MSCs is shown in the graphical abstract.

NF-jB-Stimulated IL-13 Production Also Contributes to the Increased Anti-Inflammatory Potential of iMSC-CM Over AF-MSC-CM
We then wanted to determine whether the above findings were also true for the other type of PSC-MSCs, iMSCs. We first reprogrammed amniotic fluid-derived (AF)-MSCs into iPSCs and confirmed the reactivation of the expression of pluripotency markers (Supporting Information Fig. S3A) and their expression of early lineage markers upon embryoid body differentiation (Supporting Information Fig. S3B). We then generated iMSCs using the same protocol used to derive ES-MSCs and validated their MSC-like morphology (Supporting Information Fig. S4Ai), cell surface marker expression profile (Supporting Information Fig. S1, S4Aii), and their loss of pluripotency marker expression (Supporting Information Fig. S4B).
Interestingly, similar to ES-MSCs, iMSCs exhibited an increased proliferation rate in comparison to AF-MSCs (Supporting Information Fig. S4C). Moreover, iMSC-CM exhibited increased antiinflammatory potential (Fig. 5A), which was concomitant with increased NF-jB activation (Fig. 5Bi-5Biii) and increased production  (Fig. 5Ci-5Ciii). Finally, we demonstrated that increased IL-13 production contributes to the increased anti-inflammatory potential of iMSC-CM over AF-MSC-CM by showing that an IL-13 inhibitory peptide significantly reduced the ability of iMSC-CM to reduce NF-jB activation in reporter cells (Fig. 5D).

DISCUSSION
To our knowledge, this is the first direct comparison of the therapeutic potential of PSC-MSCs and primary fetal MSCs. PSC-MSCs have been shown to have superior immunomodulatory properties to adult MSCs [43,53]; however, we hypothesized that fetal MSCs may be comparable to PSC-MSCs due to their more primitive origin. Here we show that, contrary to our hypothesis, PSC-MSCs proliferate faster than AF-MSCs and have greater antiinflammatory potential than both fetal BM-MSCs and AF-MSCs. These results suggest that PSC-MSCs are the optimal cell source for therapeutic use, even over our current "gold standard," fetal MSCs. The use of PSC-MSCs would bypass the challenge of replicative senescence, since pluripotent cells can self-renew indefinitely in culture, and using the same cell source for multiple therapies would allow a level of standardization that would not be possible with other sources of MSCs.
As mentioned, PSC-MSCs have superior therapeutic capacity in a wide variety of disease contexts, yet we lack understanding of the molecular mechanisms underlying this phenomenon and of MSC therapeutic mechanisms more generally. Here we show that PSC-MSCs exhibit increased NF-jB activation in comparison to AF-MSCs and that this occurs concomitantly with increased IL-13 production and anti-inflammatory capacity, providing for the first time a potential explanation for the increased therapeutic potential of PSC-MSCs over other sources of MSCs.
Moreover, we demonstrate the first comparison of the two different PSC-MSC types, iMSCs and ES-MSCs. We and others [57] show that iMSCs exhibit decreased levels of MSC marker expression in comparison to ES-MSCs and AF-MSCs and that iMSCs are a more heterogenous population generally. We have also observed that ES-MSCs have similar size and granularity to AF-MSCs than iMSCs do and that ES-MSCs release higher levels of the antiinflammatory cytokines IL-10 and EGF and lower levels of the proinflammatory cytokine IL-6 in comparison to iMSCs (data not shown), suggesting that ES-MSCs are the most appropriate for therapeutic use. Despite this, iMSCs present the advantages of being able to be used autologously, or to be patented, which are not possible in the case of ES-MSCs.
Our study also shows that first trimester AF-MSCs have neuroprotective potential following HI injury in the mouse brain. Our HI model mimics prenatal brain injury that can lead to lifelong cognitive disabilities such as cerebral palsy and epilepsy, and the ability of MSCs to prevent this. Our results fit with previous findings that demonstrate the ability of BM-MSCs and multipotent adult progenitor cells (MAPCs) to exert neuroprotective effects after HI in rodent and larger animal models [58][59][60][61][62][63]. Moreover, we show for the first time that ES-MSCs exhibit greater neuroprotective potential than AF-MSCs in this context. NF-jB-stimulated IL-13 production may be one of many ways in which MSCs protect the brain after HI injury. For example, NF-jB is also likely to stimulate the production of additional protective cytokines and small molecules such as IL-10, PGE2, and IDO either directly or in combination with other factors; we have observed the increased production of IL-10 by ES-MSCs in comparison to AF-MSCs (data not shown). In addition, we and others [42] show that ES-MSCs express lower levels of IL-6 in comparison to other MSC types, which may contribute to their increased therapeutic effects since IL-6 generally acts as a pro-inflammatory cytokine. We also identify IL-1ra and MIF as cytokines that are produced at higher levels by PSC-MSCs than AF-MSCs. Zhang et al. [11] have previously shown that MIF (and growth/differentiation factor 15) production plays a role in the increased cardioprotective role of iMSC-CM over BM-MSC-CM and IL-1ra has been shown to be important for the therapeutic effects of MSCs in the lung [64]. Lian et al. [44] also demonstrated increased production of stromal cell-derived factor (SDF)1a, stem cell factor (SCF) and fibroblast growth factor (FGF)2 in iMSCs compared with the parental cell population. It is likely that these cytokines are context-dependent and that cocktails of factors, rather than one individual factor, are important since the outcomes from early clinical trials with single cytokines for brain and cardiovascular diseases has failed to meet expectations [63,64]. One potential challenge is the lack of stability of growth factors, which could potentially be addressed by the use of extracellular vesicles [12,13,15].

CONCLUSION
By illuminating the molecular mechanisms underlying the improved therapeutic potential of PSC-MSCs, and therefore of MSCs more generally, our work may have implications for improving the efficiency of MSC therapies. For example, NF-jB signaling could be manipulated in MSCs using a lentiviral IKK2 overexpression plasmid to improve the therapeutic potential of the extracellular vesicles released by MSCs. The anti-inflammatory properties of PSC-MSCs are clearly superior not only to adult MSCs but also to fetal MSCs and it is, therefore, likely that they represent the most promising source for therapeutic use in the future, so long as their long-term stability is proven and risks of teratoma formation are circumvented.