Effect of Different Inoculum Densities of S. rolfsii on Seed Germination, Seedling Growth Parameters and Disease Incidence

The groundnut production in India was 71.8 lakh tons in 2015-16. There were 9 States having groundnut production of more than 1 lakh ton viz. Gujarat, Rajasthan, Tamil Nadu, Andhra Pradesh, Karnataka, Madhya Pradesh, Maharashtra, West Bengal and Telangana in 2015-16. Gujarat was the leading producer of groundnut with 28.9 lakh tons in 2015-16 followed by Rajasthan 10.41 lakh tons and Andhra Pradesh ranks 4 th leading producer in India with 7.88 lakh tons in 2015-16. It increased by 2.95 lakh tons as compared to the groundnut production of 4.93 lakh tons in the year 2014-15. Thus, the annual growth recorded in the year 2015-16 was 59.84%. (Source: Production of Groundnut from 2013-14 to 2015-16 from Ministry of Agriculture and Farmers Welfare).

Introduction
The groundnut production in India was 71.8 lakh tons in 2015-16. There were 9 States having groundnut production of more than 1 lakh ton viz. Gujarat, Rajasthan, Tamil  The low productivity of groundnut in India was attributed to several biotic and abiotic factors. Groundnut is susceptible to wide range of microorganisms which include fungi, viruses, mycoplasma, nematodes and bacteria (Khirood and Paramjit Kaur, 2013). Among them, stem rot caused by Sclerotium rolfsii Sacc. is a major problem and is an economically important soil borne pathogen. It In the present investigation effect of different inoculum densities of S. rolfsii on the development of stem rot of groundnut was assessed using soil infestation method in pot culture under green house and highest germination (45.83), root length (12.37 cm) and shoot length (15.16 cm) was observed at 1 per cent inoculum level. Germination percentage, root length and shoot length of groundnut was gradually decreased with increase of inoculum levels from 1 to 5 per cent. Lowest germination (16.66%), root length (7.50 cm), shoot length (8.33 cm) was observed in 5 per cent inoculum levels. Since the germination of groundnut seeds was reduced to more than 50 per cent at one per cent inoculums level, it was followed throughout the investigations.
causes pod yield losses of 10-25%, but under severe diseased conditions yield losses may range up to 80% (Rodriguez Kabana et al., 1975). Stem rot is a persistent soil borne disease throughout India and its incidence is increasing even at maturity stage of the groundnut crop. Though S. rolfsii survives both on seed and in soil, a greater threat is posed by soil borne inoculum (Kumar et al., 2013). S. rolfsii forms brownish sclerotia that can survive for long periods in the soil and retain their viability by tolerating biological and chemical degradation due to the presence of melanin in the outer membrane (Chet, 1975). S. rolfsii is a necrotrophic soil borne fungal pathogen, which has a very wide host range and distributed worldwide in warm climates (Aycock, 1966).
S. rolfsii was first reported on tomato by Rolfs (1892) and later the pathogen was named as Sclerotium rolfsii by Saccardo (1911). Higgiens (1927) worked in detail on physiology and parasitism of S. rolfsii. This was the first detailed and comprehensive study in USA. It is distributed in tropical and subtropical regions of the world where high temperatures prevail. The fungus has a wide host range of 500 species in about 100 families including groundnut, green bean, lima bean, onion, garden bean, pepper, potato, sweet potato, tomato and water melon (Aycock, 1966).
Stem rot of groundnut has become one of the major constraints in recent years. Management of stem rot disease is difficult because of its soil borne nature and the chemical methods are very expensive and will not be that good affect against the pathogen. In view of unsatisfactory results by chemical methods, considerable attention has been given on the other non-chemical means of plant disease control i.e., the integration of biological methods which include the use of eco-friendly bio control agents. Kilpatrick and Merkle (1967) reported the effect of different levels of S. rolfsii inoculum on the foot rot of wheat and found that, 0.5 and 1.0 per cent inoculum was superior to 3, 5 and 10 per cent level. Nargund (1981) considered two per cent inoculum level as sufficient to cause considerable infection on foot rot of wheat, even though hundred per cent disease incidence was noticed at six per cent and above inoculum level. Palakshappa et al., (1987) observed considerable foot rot infection, when betelvine were inoculated with two and three per cent inoculum and recorded 100 per cent infection at four per cent and above inoculum levels. Harlapur (1988) reported that, two per cent inoculum was essential for infection. But, maximum infection (100%) was noticed at inoculum level of more than four per cent in foot rot disease of wheat.
Mc Clain and Scharpf (1989) reported a positive correlation between seedling mortality of coniferous species and inoculum density, whereas plant height and weight of the surviving seedlings were negatively correlated with sclerotial density of Macrophomina phaseolina.
Uma Singh and Thapliyal (1998) reported inoculum density levels of 2.5 to 10 g kg -1 soil significantly increased the emergence of seed and seedling rot of soybean caused by S. rolfsii which was ranged from 36.70 to 90 per cent. Azhar et al., (2006) observed positive correlation between disease severity and inoculum concentrations (3-20 g/560 g of soil) in collar rot of chickpea incited by S. rolfsii, where seedling mortality increased with an increase in inoculum load.
The seeds of groundnut were sown after two days of pathogen inoculation @ 8 seeds per pot and an uninoculated control was maintained. The pots were observed for seed germination and per cent seed germination was recorded. After 15 days of sowing root length, shoot length, Per cent Disease Incidence (PDI) and Per cent Disease Severity were recorded. Each treatment was replicated three times.

Results and Discussion
Studies on the effect of inoculum densities of S. rolfsii on the incidence of stem rot in groundnut revealed that the germination percentage of groundnut seeds was highest at one per cent inoculum density (45.83%) i.e., 52.17 per cent reduction over control. The least germination was recorded at 5 per cent inoculum density (16.66%), i.e., 86.21 per cent decrease over control. The results are presented in the Table 1 and Figure 1. Germination of groundnut seeds gradually decreased with increase in inoculum densities from 1 to 5 per cent and was significantly different from each other. The germination percentages recorded at different inoculum densities were in the following order.
The highest root length was recorded at one percent inoculum density (12.37 cm) and the least at 5 percent inoculum density (7.50 cm). Root length of groundnut plant gradually decreased with increase of inoculum densities from 1 to 5 per cent (Plate 1). The root length at 1 per cent inoculum density was reduced to 17.69 per cent compared to control (15.03 cm) and the difference was significant. The difference in root length was significantly different from each other at 1 and 2 per cent inoculum density. The root length decreased with increase in the inoculums density however, the reduction in the root length was not significant at different densities from 2 per cent to 5 per cent. The root length recorded at different inoculums densities were in the following order.
Control > 1% > 2% > 3% > 4% > 5% Shoot length was highest at one percent inoculum density (15.16 cm) i.e., 21.85 per cent reduction over control (19.40 cm). Least shoot length was recorded at 5 percent inoculum density (8.33 cm) which is 57.61 per cent reduction over control. Shoot length of groundnut plant gradually decreased with increase of inoculum densities from 1 to 5 per cent (Plate 2). The reduction in shoot length observed at one percent inoculums density is significant compared to control however, the difference in shoot length recorded from 1 per cent to 4 per cent inoculum densities was not significant. The reduction in the shoot length was significant between 4 per cent to 5 per cent inoculum densities. The shoot length recorded at different inoculums densities were in the following order.
Control > 1% > 2% > 3% > 4% > 5% From the above experiment, it is clear that even at one percent inoculum density the germination percentage of groundnut seeds was affected by more than 50 per cent. Hence throughout the experimentation one percent inoculums was applied and also sick soil was maintained by adding one percent inoculum.
Disease symptoms were not observed in any of the treatments at 14 days after sowing. However development of sclerotial bodies were observed at 4 and 5 per cent inoculum (Plate 3). Mc Clain and Scharpf (1989) reported a positive correlation between seedling mortality and inoculum density in coniferous species, whereas plant height and weight of the surviving seedlings were negatively correlated with sclerotial density of Macrophomina phaseolina. Azhar et al., (2006) reported that seedling mortality increased with increase in inoculum levels in collar rot of chickpea. Harlapur (1988) noticed maximum infection (100%) in inoculum level of more than four per cent in foot rot disease of wheat.
Germination percentage, root length and shoot length of groundnut was gradually decreased with increase of inoculum levels from 1 to 5 per cent. Lowest germination (16.66%), root length (7.50 cm), shoot length (8.33 cm) was observed in 5 per cent inoculum levels. Since the germination of groundnut seeds was reduced to more than 50 per cent at one per cent inoculums level, it was followed throughout the investigation.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
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.


text:
Sucrose assists selection of high‐quality oocytes in pigs

Abstract We investigated whether high‐quality in vitro matured (IVM) oocytes can be distinguished from poor ones based on the morphological changes after treatment with hyperosmotic medium containing 0.2 mol/L sucrose in pigs. We hypothesize that IVM oocytes maintaining round shape have higher quality than mis‐shapened oocytes following dehydration. Oocyte quality was verified by determining embryonic developmental competence using in vitro fertilization, nuclear transfer and parthenogenetic activation. In all cases, the round oocytes had greater (p < .05) developmental competence than that of mis‐shapened oocytes in terms of blastocyst rate and total cell number in blastocysts obtained after 6 days of in vitro culture. We also confirm that round aged oocytes are higher in quality than mis‐shapened aged oocytes. In an attempt to find out why high‐quality oocytes maintain a round shape whereas poorer oocytes become mis‐shapened following sucrose treatment, we examined the arrangement of actin microfilaments and microtubules. Abnormal organization of these cytoskeletal components was higher (p < .05) in mis‐shapened oocytes compared to round oocytes after 52 hr of IVM. In conclusion, sucrose treatment helps selection of high‐quality oocytes, including aged oocytes, in pigs. Abnormal cytoskeleton arrangements partly explain for low developmental competence of mis‐shapened oocytes.

Sucrose treatment was initially used for diffusing cryoprotectants out of the embryos during the dehydration step in oocyte/embryo cryopreservation (Leibo, 1984). Sucrose was then used as a method to aid enucleation in SCNT in mice (Wang, Liu, Li, Lian, & Chen, 2001), but it also enabled the classification of oocyte quality. The poorer quality oocytes shrunk in an irregular manner, whereas goodquality oocytes shrank spherically and showed a swelling around the meiotic spindle after treatment with the medium containing sucrose at slightly different concentrations dependent on the species (Lee et al., 2014;Liu, Sung, Barber, & Yang, 2002;Wang et al., 2001).
However, the mechanism influencing oocyte quality based on the response in hyperosmotic medium is not fully understood.
In this study, we investigated whether high-quality porcine IVM oocytes, including in vitro aged oocytes, can be distinguished from the poorer ones based on the morphological changes after being treated with 0.2 mol/L sucrose. This was determined by comparing the developmental ability of the two morphological types of oocytes in vitro. We then examined their cytoskeleton which is known to determine cell shape.

| Oocyte collection and IVM
Porcine ovaries were collected from pre-pubertal cross-bred gilts (Landrace 9 Large White 9 Duroc) at a local abattoir and transported to the laboratory in Dulbecco's phosphate buffered saline (PBS; Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) at 35°C within 1 hr. Cumulusoocyte complexes (COCs) were aspirated from 3-6 mm follicles, and cultured in groups of 40-50 in 500 lL of modified North Carolina State University (NCSU)-37 medium (Petters & Wells, 1993) without oil overlay according to Kikuchi et al. (2002)  After washing, oocytes selected for the presence of the polar body were treated with 0.2 mol/L sucrose in Medium 199 supplemented with HEPES and 5% FCS (524 mOsm/L) for 5 min. Based on the changes of morphology after sucrose treatment, the oocytes were divided into two groups, termed round and mis-shapened (Figure 1a, b). After treatment with sucrose, the oocytes were washed three times in Medium 199 with HEPES plus 5% FCS to completely remove sucrose. Oocytes were then used for either IVF, SCNT or PA.

| IVF
IVF was conducted according to Kikuchi et al. (2002). The medium for IVF was a modified Pig-FM (Suzuki et al., 2002), comprising 90 mmol/L NaCl, 12 mmol/L KCl, 25 mmol/L NaHCO 3 , 0.5 mmol/L NaH 2 PO 4 , 0.5 mmol/L MgSO 4 , 10 mmol/L sodium lactate, 10 mmol/L HEPES, 8 mmol/L CaCl 2 , 2 mmol/L sodium pyruvate, 2 mmol/L caffeine, and 5 mg/mL bovine serum albumin (BSA; Fraction V, Sigma). Groups of 15-20 oocytes were transferred into 100 lL IVF droplets covered with paraffin oil. Frozen-thawed epididymal spermatozoa were pre-incubated in Medium 199 (with Earle's salts and HEPES, pH adjusted to 7.8) for 15 min. The sperm were then diluted to give a final concentration of 1 9 10 5 sperm/mL in the IVF medium droplet with oocytes. Sperm and oocytes were co-incubated for 30 min at 38.5°C in 5% CO 2 , 5% O 2 , and 90% N 2 . The oocytes and bound sperm were then transferred into fresh IVF droplets without sperm for another 2.5 hr under the same conditions.

| SCNT
The cumulus cells were removed from COCs following IVM and then collected, washed with PBS and used as donor cells for SCNT on the same day. SCNT was performed according to Akagi et al. (2008)   The oocyte-cell complex was orientated between a pair of parallel electrodes, 1 mm apart, in fusion medium comprising 0.3 mol/L mannitol, 1 mmol/L CaCl 2 , 0.1 mmol/L MgCl 2, and 0.5 mmol/L HEPES and 0.1 mg/mL BSA. A single direct current (DC) pulse of 3 kV/cm for 10 ls was applied for oocyte-cell fusion using electro cell fusion generator LF101 (Nepa Gene Co., Ltd. Chiba, Japan). Fused embryos were activated 3-4 hr after fusion in the same manner as for PA oocytes and cultured as described below.

| Parthenogenetic activation
Sucrose-treated oocytes were parthenogenetically activated by a single DC pulse of 1.5 kV/cm for 100 ls using the same mannitol buffer as for fusion in SCNT. Electrically stimulated oocytes were then cultured 3 hr in IVC-PyrLac supplemented with 5 lg/mL cytochalasin D. The oocytes were then transferred to IVC-PyrLac as described below.

| IVC
Presumptive zygotes produced by IVF, PA and SCNT were then transferred into IVC media. Two types of IVC media were prepared (Kikuchi et al., 2002). The basic IVC medium was NCSU-37 modified by the addition of 0.4% (w/v) BSA and 50 lmol/L b-mercaptoethanol. Embryos were cultured at 38.5°C under 5% CO 2 , 5% O 2 , and 90% N 2 in IVC-PyrLac (basic IVC medium with the addition of 0.17 mmol/L sodium pyruvate and 2.73 mmol/L sodium lactate) from Day 0 to Day 2, and then in IVC-Glu (basic medium supplemented instead with 5.55 mmol/L glucose) until Day 6. Day 0 was designated the day of IVF, PA or SCNT.

| Actin and tubulin staining
Staining of actin microfilaments and microtubules was performed according to Somfai et al. (2011). Briefly, the oocytes were denuded using a fine glass pipette in  supplemented with 5% FCS and 0.1% (w/v) hyaluronidase. After sucrose treatment, the round or mis-shapened oocytes were then fixed with 3.7% (w/v) paraformaldehyde for 1 hr at room temperature (RT). After washing in PBS with 0.2% (w/v) Triton X-100 (PBS-T) at RT for 30 min, the oocytes were kept in a blocking solution, comprising PBS with 20 mg/ml BSA and 150 mmol/L glycine overnight at 4°C.
After washing three times in PBS-T, the oocytes were incubated with anti-a-tubulin primary antibody (Sigma T-5168) at a dilution of 1:100 for 2 hr at 37°C in PBS-T. The oocytes were then washed twice in F I G U R E 1 Morphological changes and arrangement of actin microfilaments and microtubules in round (a and c) and mis-shapened oocytes (b and d) after treatment with sucrose at 52 hr of in vitro maturation PBS-T. Following that, they were incubated with Alexa Fluor 488-conjugated secondary antibody (A21121; Invitrogen, Tokyo, Japan) In metaphase II oocytes, normal actin microfilaments were considered to be evenly spaced beneath the plasma membrane, whereas abnormal microfilaments were discontinuously arranged or diffused (Sun et al., 2001). Normal microtubules in mature oocytes were classified as having the polar body cap integrated, whereas abnormal microtubules did not or were discontinuously arranged (Figure 1c,d) (Sun et al., 2001).

| Evaluation of total cell number in blastocysts
To evaluate the total cell number in embryos, Day 6 blastocysts were stained with Hoechst 33342. Blastocysts were washed in PBS and treated for 5 min with 99.5% ethanol supplemented with 50 lg/mL Hoechst 33342. The blastocysts were then washed in PBS supplemented with 0.3% polyvinylpyrrolidone (Sigma) and mounted in glycerol on a glass slide and covered with a cover slip. The blastocysts were gently flattened under the coverslip, so that the cells appeared in the same focal plane. Total cell numbers were counted using an epifluorescent microscope (Nikon Diaphot 200; Nikon, Tokyo, Japan).
2.9 | Experimental design 2.9.1 | Effect of sucrose treatment on further embryonic development Oocytes after 44 hr of IVM were denuded and treated either with or without 0.2 mol/L sucrose (as control group). All oocytes with a first polar body from each group were then fertilized with frozenthawed epididymal spermatozoa. The blastocyst rate and total cell number in blastocysts on Day 6 were compared between groups.
2.9.2 | Development of round and mis-shapened oocytes following IVF, SCNT, and PA After 44 hr of IVM, the oocytes were freed of cumulus and treated with 0.2 mol/L sucrose. Based on the changes in morphology after sucrose treatment, the oocytes were divided into two groups, round or mis-shapened. The oocytes were separately then used for IVF, SCNT, and PA. The blastocyst rates and total cell number in blastocysts on Day 6 were determined. In addition, the sperm penetration, monospermy, and polyspermy rates were evaluated 10 hr post-IVF and were compared between the two groups.
2.9.3 | Actin microfilament, microtubule structure, and developmental competence of round and misshapened oocytes after 44 and 52 hr of maturation After 44 and 52 hr of IVM, oocytes were denuded and treated with hyperosmotic medium containing 0.2 mol/L sucrose. The oocytes were then divided into either round or mis-shapened oocyte groups.
A number of oocytes were subjected to immuno-fluorescence staining for actin microfilaments and microtubules, whereas the remains were used for IVF, followed by IVC.

| Statistical analysis
The data on sperm penetration, monospermy, polyspermy (for IVF), blastocyst rates (expressed as percentage development of total embryos cultured), and mean cell number in blastocysts, as well as percentages of oocytes with abnormal actin microfilament and microtubules, were analyzed by one-way analysis of variance followed by Tukey test using the Statview 5 software package (SAS Institute Inc., Cary, NC, USA). Percentage data were arcsine transformed before analysis. Data are expressed as mean AE SEM. p < .05 was considered statistically significant.

| Effect of sucrose treatment on further embryonic development
There was no significant difference (p > .05) in the blastocyst formation rates, or the total cell number in blastocysts on Day 6, after IVF between the sucrose treatments (34.0% AE 5.7% and 40.9% AE 3.8%, respectively) compared to the control group (34.6% AE 6.0% and 44.7% AE 2.8%).
Similar to the results with IVF, the percentage of oocytes developing to the blastocyst stage and the total cell numbers in blastocysts on Day 6 after SCNT in the round group (16.2% AE 2.9% and 35.2% AE 2.8%, respectively) were significantly greater (p < .05) than those in the mis-shapened group (4.5% AE 2.1% and 20.0% AE 3.0%, respectively) (Figure 2b).
Consistent with the results from IVF and SCNT, both the proportion of embryos that developed into blastocysts and their total cell number on Day 6 in PA derived from the round group (39.2% AE 3.6% and 49.0% AE 2.2%, respectively) were significantly greater (p < .01 and p < .05, respectively) than those from the mis-shapened group (15.1% AE 1.9% and 41.0% AE 1.8%, respectively) ( Figure 2c).
In agreement with the results shown previously in Figure 2a, we found that the blastocyst rate and cell number in blastocysts derived from round oocytes fertilized after 44 hr of IVM (40.6% AE 3.5% and 56.6 AE 3.0, respectively) were significantly higher (p < .05) than those derived from mis-shapened oocytes fertilized after 44 hr of IVM (16.4% AE 2.2% and 37.6% AE 4.3%, respectively) ( Table 1). Although the proportion of blastocyst derived from round oocytes fertilized after 52 hr of IVM (28.6% AE 3.5%) were significantly higher (p < .05) than that derived from mis-shapened oocytes fertilized after 52 hr of IVM (3.6% AE 1.4%), cell numbers in blastocysts derived from these two groups were not significantly different (

| DISCUSSION
In the present study, we examined whether good-quality oocytes can be distinguished from low-quality ones based on simply incubating oocytes in hyperosmotic medium supplemented with 0.2 mol/L sucrose for 5 min and assessing the resulting morphology. After short incubation in hyperosmotic medium containing 0.2 mol/L sucrose, all the oocytes dehydrated and shrank in size. However, a number of oocytes still maintain a round shape, whereas others become mis-shapened or "deformed". Based on previous reports, we hypothesize that oocytes that maintain a round shape are of higher quality than oocytes having "irregular" shape after treatment with  (Lee et al., 2014). In this study, we have examined with several sucrose concentrations (data not shown) and used hyperosmotic medium containing 0.2 mol/L sucrose. According to the morphological changes, we observed a similar effect as previous studies in determining oocyte quality.
We first examined whether treating oocytes with sucrose would compromise developmental competence of porcine oocytes. There was no significant difference in the blastocyst formation rate and the total cell number in blastocysts on Day 6 after IVF between sucrose treatment and control groups. Therefore, sucrose treatment per se did not have a negative effect on embryonic developmental competence. This observation was similar to previous reports in mouse (Wang et al., 2001), bovine (Liu et al., 2002) and pigs (Lee et al., 2014).
We next examined our hypothesis that oocytes that maintain a round shape in 0.2 mol/L sucrose medium are of higher quality, whereas those oocytes that become mis-shapened are of poorer quality. The results showed that oocytes that maintained a round shape had significantly greater developmental competence than that of mis-shapened oocytes after IVF, SCNT or PA in terms of the blastocyst formation rates and the total cell number in blastocysts on Day 6. These results were similar to previous reports in mouse (Wang et al., 2001), bovine (Liu et al., 2002) and pigs (Lee et al., 2014) and confirmed our hypothesis. This study shows that sucrose treatment could help to distinguish good-quality oocytes from poorer quality, and the treatment did not interfere with further developmental competence. Therefore, this suggests that a treatment with 0.2 mol/L sucrose followed by morphological evaluation is a simple and efficient method for selection of high-quality porcine IVM oocytes.
Microfilaments and microtubules are major components of the cytoskeletal network that determine the shape and intra-cellular movement of organelles in mammalian cells and oocytes (Kim, Funahashi, Prather, Schatten, & Day, 1996;Sun & Schatten, 2006;Sun et al., 2001;Wang, Abeydeera, Prather, & Day, 2000). Moreover, both microtubule and microfilament dynamics are integrated and interact with chromosomal changes during oocyte maturation, providing a framework for chromosomal movement and cell division (Kim et al., 1996). The cytoskeleton is also an important modulator of many fertilization and post-fertilization events (Kim, Chung, & Day, 1997;Sun et al., 2001). Actin microfilaments in particular have an important role in cell division (Cao & Wang, 1990;Kim et al., 1996) and abnormal microfilament distribution has been observed in in vitro matured and fertilized oocytes (Wang, Abeydeera, Han, Prather, & Day, 1999). Based on these findings, we hypothesized  shapened oocytes was similar to that in round oocytes after 44 hr of IVM, this proportion increased dramatically and was significantly greater than that in round oocytes after 52 hr of IVM (Figure 3).
Previous studies reported that both microtubules and microfilaments might not be required for pronuclear formation (Kim et al., 1997).
This may explain why there were no differences in sperm penetration, monospermy, and polyspermy rates between round and misshapened oocytes after IVF. However, normal actin filaments are required for normal cell division (Cao & Wang, 1990;Sun et al., 2001). Thus, abnormal actin filament distribution is a possible reason for the abnormal cleavage, fragmentation and low cell numbers commonly associated with in vitro produced porcine embryos (Wang et al., 1999(Wang et al., , 2000. In addition, microtubule assembly affects pronuclear migration, syngamy and cytokinesis in zygotes (Sun et al., 2001), and is required for contractile ring formation during cleavage (Sun & Schatten, 2006). Taken together, this suggests that the abnormalities of actin microfilaments and microtubules that occur more frequently in mis-shapened oocytes compared to round oocytes at later stages of IVM may be one reason for the lower developmental competence of mis-shapened oocytes.
The remarkable increase in proportion of abnormal actin microfilaments and microtubules in mis-shapened oocytes after 52 hr of IVM led us to perform IVF and follow development of those aged oocytes. It has been widely known that in vitro aging causes various functional disorders in oocytes and in derived zygotes and embryos, including chromosomal anomalies, apoptosis, decreased fertilization rate, increased incidence of polyspermy, reduction in maturation promoting factor (reviewed in Miao, Kikuchi, Sun, & Schatten, 2009), and cytoskeletal abnormalities (Somfai et al., 2011). However, we found that, in our study, round aged oocytes have similar developmental competence compared with round oocytes matured in vitro for 44 hr in terms of development to blastocysts and total cell number in blastocysts. Unlike round aged oocytes, mis-shapened aged oocytes had lower potential to reach blastocyst stage compared with mis-shapened oocytes matured in vitro for 44 hr. This suggested that sucrose treatment might also help to select goodquality oocytes among aged oocytes. This gives another option for researchers working with aged oocytes besides reversing the aging process by controlling the activity of maturation promoting factor (Kikuchi et al., 2000) or by transferring nuclear material of an aged oocyte into the cytoplast of a fresh oocyte (reviewed in Miao et al., 2009). Nevertheless, the percentage of round oocytes = drastically dropped from 60% (160/276) after 44 hr of IVM to 28.4% (77/ 271) after 52 hr of IVM, suggesting fewer and fewer good-quality oocytes can be selected among the aged oocytes. Moreover, a good blastocyst rate alone does not guarantee normal development to term.
In conclusion, a hyperosmotic medium containing 0.2 mol/L sucrose assists the selection of high-quality oocytes, including aged oocytes, in pigs. The maintenance of a round shape after treatment with sucrose and the integrity of actin microfilaments and microtubules are criteria for high-quality porcine oocytes. However, further research on the arrangement of the cytoskeleton at later developmental time points in IVF, SCNT and PA embryos comparing round and mis-shapened oocytes is required.

ACKNOWLEDG MENTS
We would like to thank Ms. Ando, Ms. Suzuki and Ms. Nagai for technical assistance. This study was partly supported by a JSPS -MBIE/RSNZ joint bilateral research project (David Wells, Kazuhiro Kikuchi, Takashi Nagai and Thanh Quang Dang-Nguyen).


extfieldsofstudy: Medicine Biology

text:
Quantitative spatial analysis of haematopoiesis-regulating stromal cells in the bone marrow microenvironment by 3D microscopy

Sinusoidal endothelial cells and mesenchymal CXCL12-abundant reticular cells are principal bone marrow stromal components, which critically modulate haematopoiesis at various levels, including haematopoietic stem cell maintenance. These stromal subsets are thought to be scarce and function via highly specific interactions in anatomically confined niches. Yet, knowledge on their abundance, global distribution and spatial associations remains limited. Using three-dimensional quantitative microscopy we show that sinusoidal endothelial and mesenchymal reticular subsets are remarkably more abundant than estimated by conventional flow cytometry. Moreover, both cell types assemble in topologically complex networks, associate to extracellular matrix and pervade marrow tissues. Through spatial statistical methods we challenge previous models and demonstrate that even in the absence of major specific interaction forces, virtually all tissue-resident cells are invariably in physical contact with, or close proximity to, mesenchymal reticular and sinusoidal endothelial cells. We further show that basic structural features of these stromal components are preserved during ageing.

Reviewer #1 (Remarks to the Author):
Gomariz and colleagues have used the clearing method and 3D microscopy to provide imaging of CXCL12-abundant reticular cells and the bone marrow vasculature. The quality of these imaging is great and the data suggesting that the numbers of CAR cells and sinusoidal endothelial cells are much higher than previously thought is of potential interest. In some ways, it is expected but this paper has clearly quantified it very well. The downside of the study is that most of the results are confirmatory of previous studies and descriptive. In the current state, and it is unclear whether their findings are relevant to the interplay of HSCs and their niche. Specific comments are as follows: We thank the reviewer for highlighting the technical quality of our work. We do agree that our results partially confirm those presented in other studies. Nonetheless, we would like to emphasize the technical and conceptual novelty of the results presented here.
First, we outline a complex experimental and computational pipeline, which allows us to provide high-quality, detailed image-based cellular quantifications that have not been reported to date. Second, we further employ statistical models, which have not been previously used to analyse BM tissues, to understand stromal cell organization and spatial interactions in the marrow. Third, based on these quantifications, we draw biological insight on the mode of action of these cell types and their relevance on BM physiology. Fourth, we have now included additional novel data on aged bone marrow. Finally our study reveals and calls for attention to a very important issue for the research field, namely that endothelial and mesenchymal populations that have been isolated and studied to date ex vivo, in fact represent only a really minor fraction of the native in vivo BM microenvironment.

Major comments
1. It appears that different marker sets were used in experiments that compare the numbers of cells detected by 3D-QM and flow cytometric analyses. The same markers should be used in each comparison to correctly interpret the data.
Response: We agree on the importance of the point raised by the reviewer. While we understand the use of different sets of markers can in principle be of concern, we do consider that we have provided compelling evidence to demonstrate that the strategies employed for subset identification with flow cytomery (FC) and 3D-QM are in fact strictly comparable, and therefore the populations analysed are equivalent. The reason for the minor divergence in sets of markers lies in the differences in sensitivity of detection, as well as in the reagents and type of staining methods required for each technology. A detailed explanation for both subpopulations analysed is provided below. First, specific identification of CARc by FC was achieved by gating CD45 -Ter119 -CD31 -CD140b + GFP + cells in CXCL12GFP mice. The inclusion of hematopoietic (CD45/Ter119) and endothelial (CD31) antigens as negative markers in the phenotypic signature was required to allow exclusion cell subsets of these origins, which express low levels of the CXCL12-GFP transgene 2 (Quintana et al., 2015) and data not shown) and may therefore "contaminate" the CARc gate. As shown in Figure 2g, once hematopoietic and endothelial cells are gated out, the remaining GFP + cells express the mesenchymal marker CD140b. Therefore we are positive that these cells correspond to bona-fide CARc. Due to attenuated signal intensities in 3D specimens and higher detection thresholds typical of microscopy methods compared to FC, only cells expressing highest levels of CXCL12-GFP are directly visible in 3D-QM. Indeed, the lower intensity of the GFP signal in endothelial or hematopoietic cells was not observed, and therefore the use of specific markers to negatively gate out these cells could be avoided. In fact, in the initial version of the paper we showed that all of the detected GFP + cells in 3D-QM expressed CD140b (just like in FC) and displayed the prototypical fibroblastic reticular morphology of mesenchymal networks (Supplementary Figure 2b). We have now included additional data, which clearly shows that GFP + cells quantified by imaging, do not express CD45, Ter119, or CD31 cells (Supplementary Figure 2a of revised version). Collectively, the data demonstrates that CARc visualized in images are effectively CD45 -Ter119 -CD31 -CD140b + GFP + , and largely correspond to the same population as the one detected in FC analyses.
Second, for the quantification by FC of sinusoidal endothelial cells (SEC), we employed the phenotypic signature CD45 -Ter119 -CD31 + CD105 hi Sca-1 int . Here we also stained for CD45 and Ter119 to exclude all hematopoietic, including erythroid cells, from our analysis. In turn, CD31 was employed to label all endothelial cells within the non-hematopoietic CD45 -Ter119gate. In BM images, comparable identification of endothelial cells can be directly achieved through immunostaining with one single highly specific endothelial cell marker (shown in Figure 3E). Indeed, the distinct morphological features of immunostained endothelial cells and their integration in the linings of continuous vascular networks, allow to unequivocally detect these endothelial cell types and easily distinguish them from round and small hematopoietic cells without the use of CD45 and Ter119 as negative markers. As a specific vascular antigen for imaging we chose the well-established marker endomucin over CD31. We did so for two reasons. a) staining for CD31 generated a very faint signal in sinusoidal endothelium using our staining protocols. b) CD31 is expressed by multiple hematopoietic cell types in the BM including megakaryocytes, platelets, and lymphocytes, some of which reside in close contact with sinusoids. Thus, the fluorescent signal of perisinusoidal cells would physically overlap with that of adjacent endothelial cells making it virtually impossible to precisely segment boundaries of endothelial linings. In contrast, immunostaining for endomucin provided a highly specific, high-intensity and distinct signal in vascular structures, amenable to our computational analysis (we refer to Figure 3 and Supplementary Figure 3) (Kwak et al., 2016). Once endothelial linings were identified in images we further employed the exact same Sca-1 int CD105 hi signature to discern the endothelial lining of sinusoidal walls and quantify the nuclei of SECs (which were CD105 hi Sca-1 lo and endomucin + ) (Supplementary Figure 3a and b). Collectively we are confident that our strategies for identification of SECs and CARc are highly consistent between both methodologies. We hope this addresses the concerns of the reviewer and that the changes introduced enhance the clarity of the manuscript in this specific point.
2. Given that one of the major concerns in the stem cell biology is comparison of the numbers of 3 stem cells and their niche, it will add some values to this study if the authors provide the information of the number of HSCs defined by their 3D-QM.
Response: We agree that the anatomical localization of HSCs is a topic of great interest, and in fact remains one major unresolved question in hematopoiesis-related research. Conflicting results have been reported by different groups, mostly due to the significant technical challenges associated to simultaneously and reliably visualizing sufficient numbers of HSCs and interacting cell types in an intact tissue context. Among them the most prominent is most likely the fact that the complex combinations of surface markers required for identification of HSCs in FC are not easily amenable to histological sections and even less in the case of thick 3D specimens. For this reason a variety of mouse models in which individual HSCs can be identified through expression of one single fluorescent reporter have been recently generated (Acar et al., 2015;Chen et al., 2016;Gazit et al., 2014;Sawai et al., 2016). While some of these mouse strains are very useful, in our opinion only one laboratory has convincingly established methods for 3D imaging in the BM and employed them to comprehensively analyze HSC niches in a well-characterized reporter mouse strain (Acar et al., 2015). Given the high quality of our imaging strategy we definitely would like to address this type of investigation in the future, for which we are currently in the process of gaining access to some of the mouse strains generated in recent times. Considering the relevance of the question, the existing controversies, as well as the reagents, workload and time required to deliver a nuanced analysis of HSC niches, such type of in depth study would constitute a whole independent manuscript in itself. We would like to point out that our study is not intended to focus on HSC niches but rather addresses what, in our view, are very relevant questions on BM stromal cell infrastructure and organization.
3. Recent studies have revealed that peri-arteriolar cells also play a role as HSC niche. Related to the comment above, it would be important to examine the number of peri-arteriolar cells with 3D-QM.
Response: We thank the reviewer for this suggestion. Following our line of argumentation above, we consider that the specific interactions of HSCs with periarteriolar cells fall out of the topic of our study. Nevertheless, as pointed out by the reviewer, periarterial and periarteriolar microenvironments in the BM are known to harbour a distinct population of stromal cells of mesenchymal origin (periarteriolar MSCs) and have been recently proposed to constitute functionally distinct niches, not only for HSC maintenance but also for the distinct regulation of megakaryocytes Kunisaki et al., 2013;Tamura et al., 2016). Thus, in the context of our study we find it relevant to determine the size and distribution of periarterial niches. Accordingly, we have now additionally analysed the spatial arrangement of arterial and arteriolar networks with respect to the extravascular volume of BM tissues. For these experiments we employed the arterial-specific Sca-1 signal (Hooper et al., 2009;Itkin et al., 2016;Nombela Arrieta et al., 2013) to segment arteries and arterioles instead of periarteriolar stromal populations, for which no unique specific marker set has been reported. To date, labelling of certain periarteriolar 4 stromal subpopulations has been described in a variety of transgenic mouse models, including Nestin-GFP (as the NestinGFP hi population) and in NG2Cre-ERT2 and Myh11-CreERT2 mice Kunisaki et al., 2013). Nonetheless, these reporter lines do not provide the means to unambiguously identify periarteriolar populations. Furthermore, it is also unclear to what extent the subsets labelled in the different mouse strains in fact overlap. Given that by definition these cell types lie tightly adhered to arterial/arteriolar structures, we are confident that the arterial signal allows us to spatially define these niches in a very strict manner. We have included the new data in Figure 5g and 5h of the revised version of the manuscript. As expected, our results indicate that periarterial niches are much less frequent and more heterogeneously distributed than sinusoidal vessels, with only a minor fraction of the BM space (5.6%) being adjacent to these structures. As a consequence, access to and interactions with these microdomains is much more restricted than in the case of sinusoids and CARc.
4. It would be interesting to investigate the interaction of HSCs and extracellular matrix in the bone marrow with the use of the authors' technique, which might lead to provide a novel mechanism for HSC regulation.
Response: We are in full agreement that the essential roles of the ECM in the control of HSC biology and BM hematopoietic function remain largely underexplored to date. One of the main reasons for this likely lies in the fact that the detailed visualization of individual ECM fibers at sufficient resolution and in 3D had not been achieved until now. Our data supports critical and pleiotropic roles of ECM as it strongly suggests that all HSCs are in direct contact with this ubiquitous ECM network. However, whether ECM composition varies between different microdomains of the BM remains currently unknown and will certainly be instrumental to dissect out the diverse and specific roles of this network in hematopoiesis. As argued above, we believe that the functional analysis of HSC-ECM relationships clearly goes beyond the scope of the work reported here, and will for sure be addressed in the future with 3D techniques, as well as through the generation of novel mouse models deficient in ECM proteins.
Response: We thank the reviewer for the correction and we have now defined the term 3D-QM, as "three-dimensional quantitative microscopy" the first time that it is stated in the manuscript.

Reviewer #3 (Remarks to the Author):
The paper is very well written, clear and exhaustive. It contain very useful elements for investigation of 3D spatial quantitative informations to be obtained also in other contexts, where spatial quantitative distribution analysis is required. The analysis is also performed in a precise way and the figures obtained are impressive, together with the conclusions, which highlight the importance of a 3D quantitative analysis.

5
We highly appreciate the positive comments from the reviewer. In the revised manuscript we provide additional data, which we trust the reviewer will find of relevance.

Reviewer #4 (Remarks to the Author):
Gomariz et al. describe a very detailed, elegant, and highly quantitative analysis of some components of bone marrow stroma, and definitively demonstrate how flow cytometry is an inadequate technology to assess bone marrow stroma. Instead, they use confocal microscopy of thick bone sections, and quantitative image analysis to produce definitive numbers quantifying the amount of endothelial and CAR cells and the relationship between the two cell types. Overall the finding that there are more CAR and endothelial cells than flow cytometry would make one think is not really novel as it is well known that the bone marrow is very highly vascularised, and there are published images of sections from CAR reporter mice that show their thick network. However, this fact is too often overlooked and a whole publication dedicated to it is worthwhile. I recommend addressing a few points to make the approach described truly useful to the community.
We are thankful to the reviewer for the positive comments and for highlighting the significance of our findings. We have included additional data to substantiate novelty.
The authors characterize in detail endothelial cells, demonstrating that they are either sinusoidal or arteriolar, and could even distinguish transitional vessels, however they do very little to better characterize the CXCL-12 reporter. Immunohistological stainings of CXCL12 show patterns different from the thick network of GFP+ cells. The authors should include an immunostaining for CXCL12 and compare GFP expression with protein localization, as differences in the two patterns may have implications for haematopoiesis.
Response: We agree on the importance of understanding the spatial distribution of CXCL12 within intact BM. Accessibility to this chemokine in BM tissues most likely influences migration, adhesion and cellular functions, critical for the progression along multiple stages of hematopoietic development. Nonetheless, we would like to emphasize that for the purpose of our study we solely employ GFP expression as a means to visualize, quantify and analyze spatial features of a certain specific cell type (CARc), which is labelled in the CXCL12GFP mice. We do not draw any conclusions on the relative contribution of this cell type as a source of CXCL12 nor of any other hematopoietic factor. Extensive previous work by multiple laboratories has substantiated the critical roles of CARc in hematopoietic regulation and HSC maintenance through production different factors, including CXCL12 but also SCF and IL-7 (Cordeiro . Given this proven relevance of CARc, our work focuses exclusively on the spatial interactions and abundance of this cell type. Thus, to what extent the expression reporter overlaps with the presence of CXCL12 protein, or what is the specific distribution of CXCL12 (whether secreted or intracellular) in BM, despite being a fundamental question for BM physiology, is in our view not critical for our study, nor does it affect the conclusions we draw.
To still address the reviewer's request, we have attempted to detect CXCL12 protein in the BM 6 using 3D-microscopy. Unfortunately, we were thus far not successful in our approach. Also, we are unaware of any publication in which such CXCL12 staining has been convincingly achieved in histological sections, given that the soluble or ECM-bound fractions of the protein are most likely lost during tissue processing and fixation.
Paul Frenette's group recently demonstrated through intracellular staining and FC analysis, that multiple cells, other than CARc, express significant levels of CXCL12 protein . Considering that different cellular sources of CXCL12 exist in the BM, and that the concentration of this chemokine in different tissue regions will not only depend on its production but also on its secretion, the mechanisms for its presentation in cells and ECM, and the degradation of the protein, the pattern of CXCL12 distribution in BM is most likely highly complex. As pointed out by the reviewer this remains an elusive and technically challenging question to address. Importantly, we would like to clarify that in our manuscript we do not imply that CARc are the sole producers of CXCL12.
It also would be ideal if the authors could include some novel observation about bone marrow biology, for example comparing bones of control vs. one of stressed/ infected/ aged/ irradiated/ leukemic mice.
Response: We agree with the reviewer's comment that inclusion of quantitative data on the BM under stress conditions would increase the interest and enhance the biological relevance of the paper. Following this suggestion we now provide in the revised manuscript a comprehensive characterization of CARc and sinusoidal networks in the bone marrow of aged mice (20-24 months old) using 3D-QM. The new data, which are presented in Figure 7 of the revised version of the manuscript, demonstrate that the maintenance of the structural integrity of the stromal components analysed is rather robust during homeostatic ageing. Notably, the densities of CARc, the volume of sinusoids and key parameters of CARc-sinusoidal interactions are largely comparable between young and aged mice. These findings are to some extent unexpected and therefore highlight the presence of solid mechanisms for structural maintenance in the BM. We modified the introduction, results and discussion accordingly and comment on the possible implications for ageing and hematopoiesis.
In the discussion, it should be pointed out that these findings are consistent with previous ones described by the Lin, Morrison and other groups, and relevant references included.
Response: We thank the reviewer for correctly pointing this out. It was our intention to appropriately credit previous results by other groups, which were cited in the initial version of the manuscript. More specifically we had highlighted the consistency of our work with that of the Morrison lab using 3D imaging of HSCs (Acar et al., 2015). Even though different methods were employed for sample preparation, computational image-based quantification and statistical analyses, when analysed in detail, the results obtained in the referred study, are well in line with our own findings in what refers to spatial interactions of CARc and sinusoids. We have modified the text of the discussion to emphasize the consistency of our study with that of Morrison and colleagues. In addition, 7 throughout the manuscript and more specifically in the discussion, we mention and comment on recent relevant work from multiple groups on the use of imaging techniques to explore cellular content and interactions in the BM (Acar et al., 2015;Bruns et al., 2014;Coutu et al., 2017;Kunisaki et al., 2013;Mokhtari et al., 2015;Shimoto et al., 2017).
Other points: The authors describe clearly how they process the tissues and analyse the images, however very little is said about the microscopy. What excitation lasers/emission filters were used, whether adjustments for depth were made etc.
Response: We revised the Methods section accordingly and now provide a detailed description of the imaging procedures as per the reviewer's request (see subsection on Confocal Imaging). The settings for image acquisition using different objectives are also detailed in a new table in the revised version (Supplementary Table 3).
Page 8. We reasoned corresponded to SECs and AECs, respectively -SECs and AECs were swapped. Are references presented for all markers used?
Response: We thank the reviewer for pointing this out and apologize for this mistake. The terms SECs and AECs were not written in the correct order in the sentence indicated. We have now amended the text accordingly. In the revised version, we have also included new references for the markers used to identify both endothelial subsets. Specifically, in a recent manuscript Breitbach and colleagues show that E-selectin, P-Selectin and VCAM-1 are expressed at high levels in SECs than non-sinusoidal endothelial cells (Breitbach et al., 2018). This work supports classical studies, which demonstrated that homing of hematopoietic progenitor cells to the BM takes place in sinusoidal vessels expressing vascular selectins and VCAM-1 (Mazo et al., 1998;Sipkins et al., 2005). We included these references in the revised manuscript.
Please review the scale bars presented in the figures as they are not always consistent within a figure, or there may be the measure but not the bar.
Response: We thank the reviewer for pointing this out and have now revised all scale bars in the images presented to make sure that in all cases we depict the correct size and measure of scale bars.

9
The authors have been responsive to the comments. They have added interesting data on aged bone marrow. They show no significant changes in the sinusoids, but could they comment on type H vessels or arterioles? Otherwise the paper is nice! Reviewer #4 (Remarks to the Author): The authors have addressed a number of the concerns raised, and even though the overall work remains descriptive and limited to only two bone marrow microenvironment components (CXCL12GFP cells and endothelial cells), I believe the manuscripts substantiate the important point that flow cytometry analyses capture only a very small proportion of bone marrow stroma cells and should therefore be complemented by microscopy-based analyses.
My major concern is that doubts remain, and are rightly raised by the authors themselves, on the nature of the GFP+ cells in the CXCL12 reporter mice used. If GFP reports CXCL12 expression, why are the authors sure that other cells produce CXCL12 too? Conversely, how can one be sure that all GFP+ cells do indeed produce high levels of CXCL12? I don't think this issue is a fundamental limitation, but I would expect the authors to discuss it somewhere in the manuscript, for example in the introduction where they motivate their studies and choice of reporters.
The analysis of bone marrow sinusoids in aged bone marrow is novel and very interesting, and deserves to be given full justice. For example, the Adams group has described in detail how endosteal vessels are affected by ageing, but these studies are not referenced in relation to the sinusoid work presented here. Together, these analyses suggest that more work will be needed to understand how space-limited changes in the microenvironment relate to dramatic changes in HSC function in aged animals. Again this point should be discussed, so that the presented work is fully put within context. 1

Reviewer #1 (Remarks to the Author):
The authors have been responsive to the comments. They have added interesting data on aged bone marrow. They show no significant changes in the sinusoids, but could they comment on type H vessels or arterioles? Otherwise the paper is nice! Response: We appreciate the encouraging feedback and thank the reviewer for the positive comments. Regarding the question on type H vessels and arterioles, we did perform stainings that allowed us to visualize arterial and transitional vessel networks in the bone marrow of old mice. While visual inspection did suggest alterations of transitional vessels, the effects were subtle and hard to assess qualitatively. Unfortunately we do not have yet the tools for the automatic and highly specific classification and quantification of transitional vessels in 3D, so as to perform a nuanced and rigorous analysis, which would allow us to draw robust conclusions on the age-induced effects on these structures. We are currently working on the development of such computational tools, which we hope to use in the near future to address these and other pertinent questions.

Reviewer #4 (Remarks to the Author):
The authors have addressed a number of the concerns raised, and even though the overall work remains descriptive and limited to only two bone marrow microenvironment components (CXCL12GFP cells and endothelial cells), I believe the manuscripts substantiate the important point that flow cytometry analyses capture only a very small proportion of bone marrow stroma cells and should therefore be complemented by microscopy-based analyses.
Response: We are thankful to the reviewer for highlighting the relevance of our findings.
My major concern is that doubts remain, and are rightly raised by the authors themselves, on the nature of the GFP+ cells in the CXCL12 reporter mice used. If GFP reports CXCL12 expression, why are the authors sure that other cells produce CXCL12 too? Conversely, how can one be sure that all GFP+ cells do indeed produce high levels of CXCL12? I don't think this issue is a fundamental limitation, but I would expect the authors to discuss it somewhere in the manuscript, for example in the introduction where they motivate their studies and choice of reporters.
Response: We understand the reviewer's point and fully agree that currently it still remains unclear whether all bone marrow CXCL12-expressing cells are in fact faithfully labelled in CXCL12-GFP reporter mice. Our statement on the fact that most likely cells other than CARc express CXCL12 is based on a recent report from the Frenette group, mentioned previously, which suggests that GFP expression in these mice does not perfectly match cellular expression of CXCL12 at the protein level . These discrepancies are to some extent puzzling and point to a complex regulation of CXCL12 expression, presentation and secretion in different BM cell types.
However, what has been so far indeed well-established in the literature and is confirmed by our 2 own studies and observations is that: i) the so-called CXCL12-abundant reticular cell population, which is GFP bright in CXCL12GFP reporter mice and LepR+, is a major source of CXCL12 in the bone marrow ii) deletion of CXCL12 expression in this subset causes major effects in hematopoiesis, including in the HSC progenitor population  iii) beyond CXCL12, CARc are the fundamental source of other critical factors, such as SCF and IL7 (Cordeiro  iv) both SECs and AECs express lower levels of CXCL12 than CARc, are thus labelled with very low/negligible GFP intensity by the CXCL12 reporter mice, which is barely detectable by confocal microscopy v) CXCL12GFP reporter mice are an optimal model to specifically visualize and study CARc.
Consequently, given that in our study we do not employ the reporter as a measurement of CXCL12 production but rather, exclusively as a marker of a relevant stromal cell population, the potential issues mentioned do not affect in any way our conclusions. In order to better clarify this point in our manuscript and following the reviewer's suggestion we have now edited the text in the Introduction and Results sections (changes highlighted in red): Introduction: Although other mesenchymal stromal cells, which are not equally labelled in CXCL12 reporter mice, have been described to produce CXCL12 , CARc are essential to marrow function as they are additionally major sources of SCF and the pro-lymphoid cytokine interleukin-7 (IL-7) Results: Hence, as previously reported, GFP bright cells in Cxcl12-Gfp mice correspond exclusively to CD45 -Ter119 -CD31 -CD140b + CARc (Omatsu et al., 2010), making this reporter strain the ideal model to specifically visualize and study this cell type via microscopy.
The analysis of bone marrow sinusoids in aged bone marrow is novel and very interesting, and deserves to be given full justice. For example, the Adams group has described in detail how endosteal vessels are affected by ageing, but these studies are not referenced in relation to the sinusoid work presented here. Together, these analyses suggest that more work will be needed to understand how space-limited changes in the microenvironment relate to dramatic changes in HSC function in aged animals. Again this point should be discussed, so that the presented work is fully put within context. Response: We thank the reviewer for underlining the novelty of the new data on aged bone marrow, which we added in the last revision. We would like to clarify that the work from Kusumbe and colleagues (Adams group) was appropriately referenced and discussed in both the Introduction and Discussion of the previous version of the manuscript. The parts of the text where this was done are shown below: Introduction: "Age-related disturbances in BM microenvironmental components are hypothesized to contribute to alterations in hematopoietic competence (Kovtonyuk et al., 2016) and include increased adipocyte infiltration (Ambrosi et al., 2017), perturbations of the cytokine milieu and reductions in bone volume and type H vessels .
3 Discussion: "In contrast to the reported age-induced loss of type H vessels , sinusoidal structures remain largely intact in the different BM regions and intrasinusoidal volumes were unchanged." Following the reviewers suggestion we have modified one sentence of the discussion to reflect the importance of addressing in future studies how changes in BM microenvironment relate to HSC dysfunction during ageing.
"Future analyses should determine whether and to what extent the aged-induced perturbations of HSC function and hematopoiesis (Kovtonyuk et al., 2016) relate to spatial and structural changes in BM organization."