Poecilia reticulata) is an ideal model for studying environmental estrogens, and its large caudal fin has a high capacity to regenerate. This study analyzed the feasibility of caudal fin for detecting vitellogenin (Vtg), the most commonly used biomarker of environmental estrogens. Firstly, a sandwich ELISA for guppy Vtg was developed using purified lipovitellin and its antibody and it had a working range of 7.8–1000 ng/mL and detection limit of 3.1 ng/mL. The ELISA was used to detect tissue distribution of Vtg. In male guppy exposed to 50 and 100 ng/L 17β-estradiol (E2), Vtg concentration in caudal fin was higher than that in whole fish, brain, eyes, gonad, and skin, and was close to that in the liver. Furthermore, male guppies were exposed to environmental concentrations of 17a-ethinylestradiol (EE2) and bisphenol S (BPS) to validate the utility of caudal fin Vtg for detecting estrogenic activities. The lowest observed effect concentration of EE2 and BPS were lower than 2 ng/L and 1 μg/L, which were below or equal to the values reported for other species, demonstrating that caudal fin Vtg was highly sensitive to estrogenic chemicals. Therefore, caudal fins of guppies are suggested as alternative samples for Vtg biomarker detection.Guppy ( To screen chemicals with estrogenic activity, US Environmental Protection Agency and Organization for Economic Cooperation Development (OECD) have developed test guidelines using small fish species as model organisms and vitellogenin (Vtg) as a core biomarker5. Currently, plasma and whole-body homogenates (WBH) are normally used for Vtg detection, which involves killing experimental organisms7. In some cases, such as field sampling of natural populations and serial monitoring of the effects of estrogenic chemicals on experimental organisms, a less invasive sampling method for repeated Vtg measurements is needed. Several studies reported that skin mucus was an alternative choice for Vtg quantification9. However, Maltais et al.10 and our previous study11 found that Vtg levels in fish skin mucus were considerably lower than those in plasma, demonstrating that surface mucus Vtg induction exhibited a lower sensitivity to estrogenic chemicals. Recently, Zhong et al.12 reported that Vtg could be detected in various extrahepatic tissues of 17α-ethinylestradiol (EE2)-exposed male zebrafish (Danio rerio), and recommended skin and eye tissues as for Vtg analysis. Sampling of these tissues would kill the fish, thus it was unable to measure Vtg induction at a later point in time. By contrast, caudal fin has a high capacity to regenerate, and zebrafish caudal fin could quickly regenerate in approximately two weeks after 95% excision13. We speculated that using caudal fins as the sampling tissue could be helpful for continuous measurement of Vtg biomarker. However, it is still unclear whether caudal fin contains Vtg and whether the sensitivity of Vtg induction in caudal fin is high enough to detect weak estrogenic activity.Environmental estrogens have aroused great concern worldwide owing to their adverse effects on wildlife health, including altered sex hormone levelsPoecilia reticulata) has a large caudal fin, which is almost half of their total length and can regenerate quickly and reliably after amputation, restoring both size and shape15. Thus, it provides a good model to evaluate the feasibility of caudal fin Vtg as a less invasive biomarker for detecting environmental estrogens. Guppy is easily cultured in the laboratory and easy to identify gender due to its obvious secondary sexual characteristics  were measured by Western blot and the sandwich ELISA to evaluate the possibility of using caudal fin for Vtg detection. Furthermore, the reliability of Vtg in caudal fin as a biomarker of estrogenic contamination was validated by quantifying Vtg levels in WBH, liver, and caudal fin of male guppy exposed to environmental concentrations of two different exogenous estrogens, EE2 and bisphenol S (BPS), which are two commonly detected estrogenic chemicals in aquatic environments.In the present study, guppy lipovitellin (Lv), the main egg yolk protein derived from Vtg, was purified and used to prepare antibodies. Using the purified Lv and its antibody, a sandwich ELISA for measuring guppy Vtg was established. Subsequently, Vtg induction in the WBH, caudal fin, liver, brain, eye, gonad, and skin tissues of male guppy exposed to different concentrations of 17Ovarian homogenate yielded two peaks in gel filtration chromatography and the first peak appeared clear protein bands Fig. . Thus frThe purified protein was stained positively with Schiff reagent, methyl green, and Sudan black B Fig. , confirmThe apparent molecular weight of guppy Lv was estimated to be approximately 480 kDa by native-PAGE Fig. . On SDS-2-exposed male guppy and the purified Lv, while no visible bands were detected in WBH from male fish , and the detection limit was estimated as 3.1 ng/mL . For 50 ng/L E2 group, Vtg concentrations in caudal fin, whole fish and liver were 1.31 ± 0.10 μg/g, 0.71 ± 0.06 μg/g, and 1.56 ± 0.15 μg/g. Moreover, Vtg concentration in caudal fin was significantly higher than in the whole fish (P < 0.05).Vtg concentrations in each tissue and WBH of male guppy in control and exposure groups were quantified by ELISA Fig. . In cont2 for 21 days were 0.35 ± 0.08 μg/g, 0.55 ± 0.08 μg/g, and 2.18 ± 0.37 μg/g, respectively 26 and rare minnow (Gobiocypris rarus)27. In order to avoid the matrix effect, all tissues except caudal fin were diluted at least 1:40 in the routine assay. The average intra-assay and inter-assay CVs of Lv-based sandwich ELISA were 2.1% and 4.1%, respectively, which were lower than those of ELISAs developed using Vtg as antigen29, demonstrating that the established ELISA had high precision. Moreover, the parallelism observed between the Lv standard curve and WBH dilution curves of E2-exposed male guppy demonstrated that anti-Lv antibody recognized Lv and Vtg similarly. In addition, the ELISA did not detect positive signal in control male WBH, indicating that this assay was very specific to Vtg31. The above results confirmed that the sandwich ELISA established in the present study could accurately quantify guppy Vtg.In the present study, Vtg in caudal fin of guppy was found to be highly sensitive to exogenous estrogens, and it could be used as a less invasive biomarker for measuring estrogenic activity. As the most commonly used biomarker for environmental estrogens, Vtg was normally quantified by ELISA33. Male guppies have a large caudal fin that is easily regenerated15. If the sensitivity of Vtg induction in caudal fin is close to that in WBH, caudal fin could replace the whole fish as the preferred sample for Vtg detection, which will be more suitable for field investigations of wild populations and for repeated vtg measurements. In the present study, Vtg concentrations in caudal fin, liver, brain, eyes, gonads, skin of male guppy exposed to E2 were quantified using the developed sandwich ELISA developed. It was found that exposure to 50, 100, and 200 ng/L E2 significantly increased Vtg concentrations in all tissues. The amount of Vtg in all exposure groups was greatest in the liver tissue, which was consistent with the tissue distribution of Vtg reported in zebrafish12. In the 100 ng/L E2-exposed group, the concentration of Vtg in caudal fin was significantly higher than that in eyes, skin, other tissues, and the whole fish. Vtg concentration in caudal fin of male fish exposed to 50 ng/L E2 was 1.85 times that of whole fish, and it was much closer to Vtg concentration in the liver than that in the 100 ng/L E2-exposed group. Evidently, Vtg induction in caudal fin had equal or even higher sensitivity than Vtg in whole fish at lower E2 concentrations (<100 ng/L). Owing to the ease of sampling and lower damage to fish, caudal fins can be good alternatives to WBH for the detection of Vtg in guppy.Guppy is a new promising model organism for studying environmental estrogens2 is the most detected synthetic estrogen that is not broken down in surface and sewage waters. Concentrations of EE2 in surface water of the UK, Japan, and Canada usually ranged from 1 to 20 ng/L36, although a maximum of 273 ng/L was found in US streams37. Many studies have reported that Vtg induction in various fish species after waterborne exposure to EE2 , and fathead minnow 40, except one study reported the LOEC of Vtg induction in male fathead minnow for EE2 was 1 ng/L41. For three-spined stickleback (Gasterosteus aculeatus), Sheepshead minnow (Cyprinodon variegatus), and Mummichog (Fundulus heteroclitus), the LOEC of Vtg induction for EE2 was higher than 50 ng/L44. In the present study, we found that 2 ng/L EE2 exposure significantly induced caudal fin Vtg, indicating that the LOEC of Vtg induction in guppy caudal fin was lower than 2 ng/L EE2, which was lower than the values reported for most tested species. Moreover, Vtg concentration in caudal fin was not significantly different from that in the liver and whole fish. Thus, the results demonstrated that Vtg in caudal fin of male guppy could detect weak estrogenic activity of EE2. In addition, this study tested the sensitivity of guppy caudal fin Vtg to BPS, which has emerged as a potential bisphenol A replacement and has been frequently detected in aquatic environments of various countries in recent years. A maximum of 7.2 μg/L of BPS was found in Adyar River of India45. Naderi et al.46 reported that 10 μg/L BPS exposure could significantly increase the plasma Vtg concentration in male zebrafish. In this study, 1 μg/L BPS exposure for 21 days was found to significantly increase Vtg concentration in caudal fin, indicating that caudal fin Vtg in male guppy had an equal or even higher sensitivity to BPS than plasma Vtg in zebrafish. The above results confirmed that Vtg induction in caudal fin of guppy was highly sensitive to estrogenic chemicals and could be used as a reliable biomarker for the detection of estrogenic activity in the aquatic environment, though the stress of handling fish repeatedly should then be taken into account.To further test the reliability of caudal fin Vtg for the detection of estrogenic activity, the present study used two common exogenous estrogens to carry out exposure experiments. EE2 concentration. Further, we found that caudal fin Vtg of guppy was capable of detecting estrogenic activities of EE2 and BPS at environmentally relevant concentrations. Therefore, we suggest guppy caudal fin with a high capacity to regenerate as a potential alternative sample for repeated Vtg analysis.In summary, a sandwich ELISA for accurate quantification of guppy Vtg was established using purified Lv and its polyclonal antibody. Subsequently, this ELISA was used to detect tissue distribution of Vtg in male guppy exposed to exogenous estrogens. Caudal fin produced Vtg in response to low concentrations of exogenous estrogens, and its Vtg concentration was significantly higher than that in the whole fish at low EPoecilia reticulata)  were mainatined in 50-L aquaria filled with 30-L dechlorinated tap water at 26 ± 1 °C, with 7.0 ± 0.1 mg/L dissolved oxygen and 14 h:10 h light-dark cycle. The fish were fed with newly hatched brine shrimp twice and half of the aquaria water was replaced daily. In addition, the fish were handled according to the National Institute of Health Guidelines for the handling and care of experimental animals and the animal utilization protocol was approved by the Institutional Animal Care and Use Committee of the Ocean University of China.Sexually mature red albino guppies  and anion-exchange chromatography  according to our previously reported methods62. After determination of protein concentration by the Bradford assay using bovine serum albumin (BSA) as the standard, the purified Lv was stored at −80 °C.Ovaries were removed from adult female guppies and homogenized in ice-cold homogenate buffer  using a glass homogenizer. After centrifugation at 8,000 × et al.63.The purified protein was analyzed using native polyacrylamide gel electrophoresis , and the gels were stained with Coomassie Brilliant Blue (CBB), Schiff reagent, methyl green, and Sudan black B following the methods as describe by Pan 64. Polypeptide components of Lv were analyzed by sodium dodecyl sulfate (SDS)-PAGE. After electrophoresis, gels were stained with CBB, and molecular weight of polypeptide units was estimated using an unstained protein ladder .The molecular mass of purified Lv was estimated by HMW native marker Kit  according to the method described by Sun and Zhang25.Polyclonal antiserum against guppy Lv was raised in rabbits following routine methods. Briefly, rabbits were injected subcutaneously with 1 mL of Lv solution (800 μg) emulsified in complete Freund’s adjuvant followed by another three boosts of Lv solution (500 μg) in incomplete Freund’s adjuvant at 2-week intervals. Blood was collected, centrifuged, and anti-Lv immunoglobulin G (IgG) was purified from the supernatant by affinity chromatography on a HiTrap Protein G column . The purified IgG was labeled with horseradish peroxidase  by an improved sodium periodate-oxidation method2-exposed male guppy, and the purified Lv were electrophoresed on SDS-PAGE and then transferred onto polyvinylidene difluoride membranes. After incubation with anti-Lv antibody at a 1:1000 dilution, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG  at a 1:2000 dilution. Finally, the membranes were visualized with freshly prepared DAB substrate.Western blot analysis was performed to check the specificity of anti-Lv antibody to guppy Vtg. The WBH of control male and Eet al.65 with minor modification. Microtiter plates  were coated with 100 μL of anti-Lv IgG diluted in 0.05 M sodium carbonate overnight at 4 °C and washed three time with 200 μL/well of PBST (10 mM PBS containing 0.05% Tween-20). The wells were then incubated with 200 μL PBST containing 1% BSA for 1 h at 37 °C. After three washes with PBST, 100 μL of samples and standard serially diluted with PBST were added to each well and incubated at 37 °C for 1 h. The wells were washed five times and received 100 μL/well of HRP-labeled anti-Lv antibody at serial dilutions . After incubation at 37 °C for 1 h, the color was developed with 100 μL of tetramethylbenzidine enzyme substrate  and stopped by adding 100 μL of 2 M sulfuric acid. Absorbance values were measured at 450 nm in a plate reader. Standards and samples were run in duplicate.A sandwich ELISA was developed by the procedure of Mitsui 30. Briefly, precision was evaluated using various purified Lv concentrations by measuring the intra- and inter-assay coefficients of variation (CV%), which were defined as the standard deviation devided by the mean and mutiplied by 100. The specificity was assessed by comparing curves of serial dilutions of WBH from E2-treated male guppy and the Lv standard curve. The limit of detection was defined as the concentration corresponding to the mean of the absorbance values for 12 replicates of the zero standards plus two times the standard deviation. Additionally, the matrix effect of caudal fin and WBH samples were evaluated by two different methods38.The reliability of ELISA assay were evaluated by measuring its precision, sensitivity, and specificity2 in 5-L aquaria. The stock solution of E2 was prepared in ethanol and kept at 4 °C. The ethanol concentration in each group was below 0.001%. The exposure solution was renewed daily. After 21 days of exposure, fish were anesthetized in a bath of tricane methane sulfate, body lengths and weights were measured. The liver, eyes, testis, brain, and skin of each fish were separated with sterilized scissors and tweezers. Stainless steel blades were used to cut one third of caudal fins with an approximate weight of 0.02 g. Each tissue was weighed, diluted 1:4 (v/v) in 10 mM PBS containing 0.02% aprotinin, and homogenized by an automatic grinding machine  with a homogeneous velocity of 50 HZ for 2 min. After centrifugation  at 4 °C, the supernatant was transferred to a clean tube for Vtg detection by Western blot and sandwich ELISA assay. All assays were carried out in duplicate.Adult male guppies (n = 16) were exposed to nominal concentrations of 50, 100, and 200 ng/L E2 and 1, 10, 100 μg/L BPS, respectively. After 21 days, caudal fin, liver tissue, and whole body were homogenated and centrifugated as described above. Vtg in the supernatant were quantified by the developed sandwich ELISA.Adult male guppies (n = 20) were exposed to 2, 10, and 50 ng/L EEP < 0.05.Vtg induction data are presented as the mean ± standard deviation, and the differences between the control and exposed groups were assessed by one-way analysis of variance followed by a Tukey’s post hoc tests. Prior to parametric analysis, data were log-transformed to achieve variance homogeneity. All statistical tests were conducted using SPSS 18.0 software , and values were determined as significant when All data has been included in the manuscript. The primary data is available to all interested researchers by contacting wangjun@ouc.edu.cn.Supplementary information


Eumetopias jubatus) pups individually marked at four of five rookeries in southeastern Alaska (SEAK) and resighted for 21 years to examine senescence, annual variability and covariation among life-history traits in this long-lived, sexually dimorphic pinniped. Sexes differed in age of onset , but not rate  of senescence. Survival of adult males from northern SEAK had greatest annual variability (approx. ±0.30 among years), whereas survival of adult females ranged approximately ±0.10 annually. Positive covariation between male survival and reproductive success was observed. Survival of territorial males was 0.20 higher than that of non-territorial males, resulting in the majority of males alive at oldest ages being territorial.Population dynamics of long-lived vertebrates depend critically on adult survival, yet factors affecting survival and covariation between survival and other vital rates in adults remain poorly examined for many taxonomic groups of long-lived mammals . We used mark–recapture models and data from 2795 Steller sea lion ( In longSenescence is within-individual physiological deterioration with age, a principal cause of which is believed to be cumulative damage to biomolecules caused by oxidative stress . SenesceActuarial senescence  is common in wild populations of vertebrates ,16. ActuIn addition to intrinsic factors, sex differences in senescence are expected to be more pronounced in polygynous than monogamous species, with rate of male senescence expected to increase with sexual selection among males . These eEumetopias jubatus), occurring from California north around the Pacific Rim and into the Bering Sea, are polygynous, highly sexually dimorphic, and the most massive sea lion species S models sults of  which folts from . Model 3lts from .p models was the best p model from [p differed from ages 1–3 but was similar at ages 4–8, and suspected potential reduced p at older ages when S may also decline [a: 1–3 separate, 4–14, 15+; b: 1–3 separate, 4+). We fit four additional structures for males which showed a more complex age pattern to age 8 than females . In addition to age categories, we also reconsidered effects of nr and nr × year on p given later adult ages were now available. Potential models with nr effects included: (1) additive nr effect the same for all ages; (2) additive nr effect only for adult females; (3) additive nr effect for adults both sexes; (4) nr × year effect the same for all ages; (5) nr × year effect differing from juveniles and adults; and (6) nr × year effect only for adult females. For models including nr effects, we considered three categories for nr: (1) all separate F, H, W and G; (2) F, H, WG pooled  and als females  (a: 1–8 s at W/G ); and , eight models for males , and four for animals aged 3+ sexes pooled . We pooled sites into north (WG) and south (FH) because dynamics differ between north and south and are similar within north and south [S of juveniles was higher for animals born in the north than in the south, and nearly identical for animals born at F and H [S of males may occur [Annual variation in  effects ,33,35) wnd south . For exa F and H . Pooling2.3.Annual capture histories for males pooled multiple sightings from May to September in a year into a single sighting by best location × territorial ‘state’ in that year. Instead of coding 1 (seen) and 0 (not seen) used for CJS models, for MS models, ‘1’ was replaced by states A , B , C , N , T  or H . Best state for that year was determined as N > H; all other codes were exclusive. The informative comparison was states A/B/C versus N, with a control for T/H, because territorial behaviour recorded at rookeries other than F was not useful due to only brief surveys after 21 June at other rookeries and haul-outs . Males from the 1994–1995 cohorts were of breeding age before 2007, but were considered of unknown state before 2007 due to insufficient survey effort. A few males from the 1994–1995 cohorts remained by 2007; the 2001–2005 cohorts contributed the most data.S and p models was the best model for males from §2.1, with additive effect of state also in p. A single p model was fit because we expected p varied by state due to high survey effort at F and to higher p of territorial versus non-territorial males. We first modelled ψ using a base model state × to state × nr3 × age, where nr3 was natal rookeries: F, H and pooled WG . Age was included as: ‘age3’ , ‘age2’  or no age effect. ψ was fixed to 0 for ages 0–4 as males were not territorial at these ages. The best ψ model was then fit to 7 S models which pooled states in S. Because fitting effects for A, B and C separately was not possible due to small sample sizes of these states, modelling was done separately for three datasets in which ABC was pooled various ways with N. Finally, we considered The basis for 3.3.1.a); thus age-specific S for males could not be estimated after age 14 , and we fit a B-spline only to ages 0–14 for males with S at age 15+ pooled as a separate parameter. A Less than 6 males remained after age 14, compared to 44–22 females per age from ages 14–20 a; thus ap model was age × sex + nr (adults only) + year . Age as categories was preferred, but because no single best age structure was apparent (QAICc weight of top five models ranged 0.06–0.11), we used the most complex structure occurring in the top five models  for modelling S. Categorical age was also strongly supported for S , but the S model with age fit using B-splines with a few less parameters was most preferred . a, electronic supplementary material, table S3). Senescence in S for F females was most apparent after age 17 with a). a). Based on the B-splines model, male a). Using the S model with categorical age (model 2), senescence in S for both sexes was strongly supported .The best 3.2.S was statistically supported only for northern-born males aged 3+ . Overall annual variation in  F and W .Figure 3.3.Seventy-one males were territorial at rookeries other than F from 2000–2015, including two at Biali Rocks, 22 at H, 18 at G, 28 at W and 1 at Sugarloaf Island . All helt age 10 . S^  was4.S of females from SEAK had strong statistical support. Although b) in SEAK and Russia, respectively [S from [Senescence in ectively . Assuminr S from  to inclu–30 (see ). This mb). It is possible rate of male senescence was higher in Russia than in SEAK with average rates of decline in S per age of −0.080 (after age 11) and −0.046 (after age 8), respectively . Small sample sizes of older ages as well as few cohorts marked per area (two in SEAK and one in Russia) prevent a true comparison of area-specific patterns. Owing to low overall survival and earlier onset of senescence, but perhaps not a greater rate of senescence, the lifespan of male SSL is particularly short. Similarly, in wild boar (Sus scrofa), male mortality rates overall were higher than those of females, but rates of senescence were similar for both sexes [Ovis aries), parasite burden increased linearly with age in males but non-linearly in females, affecting only oldest females [Onset of male senescence was earlier in our study ages 8–9) than in Russia (approx. ages 10–11) b. It is th sexes . Both agth sexes . Delayed than in  females . Because females .Mirounga angustirostris, ages 16–21 but sample sizes were less than 15 individuals after age 8 [Monachus shauinslandi, ages 17–18 [Phocarctos hookeri, Arctocephalus tropicalis) than SSL [Zalophus californianus) born in Mexico was detected at ages 10+ (c). Actuarial senescence was not detected in females to age 20 in Antarctic fur seals  [Mirounga leonina) [Leptonychotes weddellii) [Patterns of senescence observed in other pinniped species to date are diverse, but studies are few and many have limited sample sizes of older ages . Age of onset of senescence in female SSL was similar to northern elephant seals (er age 8 ), Hawaiies 17–18 ), and Ney age 20 ). Onset than SSL . Also inthan SSL . Senescetermined . A uniqutermined . Senescetermined  (figure  age 14) , nor in leonina) ,70, and ddellii) ,72 despid). Similarly, for this species in Mexico, Survival patterns of male SSL were most similar to those of California sea lions . y age 12  , suggesting this demographic group may be the most sensitive indicator of environmental conditions, if this pattern is due to prey availability rather than other causes. Male survival, in general, may be more condition-dependent than female survival . This maUnlike Hawaiian monk seals in which temporal variation in adult survival was similar among sexes , none ofNo territorial males established territories at more than one rookery during our study, suggesting breeding rookery philopatry was approximately 1.0. In contrast, natal philopatry ranged 0.60–1.0 , but obsDama dama, bighorn ewes Ovis canadensis, and possibly older male northern elephant seals [We observed positive covariation between survival and territoriality in males . This cont seals ,42,78. Ant seals , the lik


Fibroblast growth factor‐2 (FGF‐2) is a key hMSC mitogen often supplemented to increase hMSC growth rates. However, hMSCs also produce endogenous FGF‐2, which critically interacts with cell surface heparan sulfate (HS). We assessed the interplay of FGF‐2 with a heparan sulfate variant (HS8) engineered to bind FGF‐2 and potentiate its activity. Bone marrow‐derived hMSCs were screened in perfused microbioreactor arrays (MBAs), showing that HS8 (50 μg/ml) increased hMSC proliferation and cell number after 3 days, with an effect equivalent to FGF‐2 (50 ng/ml). In combination, the effects of HS8 and FGF‐2 were additive. Differential cell responses, from upstream to downstream culture chambers under constant flow of media in the MBA, provided insights into modulation of FGF‐2 transport by HS8. HS8 treatment induced proliferation mainly in the downstream chambers, suggesting a requirement for endogenous FGF‐2 accumulation, whereas responses to FGF‐2 occurred primarily in the upstream chambers. Adding HS8 along with FGF‐2, however, maximized the range of FGF‐2 effectiveness. Measurements of FGF‐2 in static cultures then revealed that this was because HS8 caused increased endogenous FGF‐2 production and liberated FGF‐2 from the cell surface into the supernatant. HS8 also sustained levels of supplemented FGF‐2 available over 3 days. These results suggest HS8 enhances hMSC proliferation and expansion by leveraging endogenous FGF‐2 production and maximizing the effect of supplemented FGF‐2. This is an exciting strategy for cost‐effective expansion of hMSCs. S Clinical use of stem cells will require effective methods to expand stem cell numbers. This work used a purpose‐engineered carbohydrate to target a key growth factor pathway that facilitates stem cell expansion, which is potentially a cost‐effective alternative to current expansion techniques. To understand the interplay of the carbohydrate with the growth factor, and its effects on stem cells, a microbioreactor array—a “lab‐on‐a‐chip” system—was used to assess many different conditions in parallel, streamlining the process of optimizing the culture conditions and understanding the mechanism of action of the carbohydrate.Human mesenchymal stem/stromal cells (hMSCs) Human MSCs are known to produce a plethora of growth factors at varying levels under normal expansion conditions Endogenous FGF‐2 secretion is dependent on native heparan sulfate (HS) chains proximal to the cell membrane Exogenously supplemented FGF‐2, on the other hand, is known to be unstable due to thermal aggregation and thermal and enzymatic degradation. FGF‐2 bioactivity decays and it loses the ability to cause phosphorylation of extracellular signal‐regulated kinase (ERK) after 24‐hours incubation in medium at 37°C, yet this can be rescued by binding of heparin to FGF‐2 protein We have previously shown that supplementation of hMSC cultures with the generic hyper‐sulfated glycosaminoglycan (GAG) heparin, results in adverse changes in the biological properties of the cells To explore the utility of HS8 for hMSC expansion, we used MBAs to screen combinations of FGF‐2 and HS8 on hMSCs and dissected the interactions between HS8 and endogenous and exogenous FGF‐2. The MBA platform is a microfluidic cell culture‐screening array providing both combinatorial mixing of factors, and continual perfusion of culture medium through serially connected culture chambers. In this device, progressive accumulation of cell secretions from upstream to downstream chambers introduces paracrine factor effects, enabling not only a rapid manner by which to search for preferential combinations of exogenous factors, but potentially the elucidation of difficult to detect paracrine‐dependent cell responses. Deployment of the MBA is intended to save on time, biological materials, and manual errors, while increasing the landscape of cell culture environments that can be rapidly generated and compared side‐by‐side, with the end goal of utilizing it as a rapid development and QC tool for cell therapies. With this approach, we gain insight into the interplay between FGF‐2 and HS8 and the effects on hMSC growth.http://www.sigmaaldrich.com), and all cell culture and detection reagents from Life Technologies , unless otherwise mentioned. FGF‐2 was purchased from R&D Systems . The engineering of HS8, including isolation, biochemical characterization, and evaluation of biological activity, including on hMSCs, was recently published by our group All fine chemicals were obtained from Sigma Aldrich  was perfused through individual factor channels, with all other inlets containing MilliQ water, at 1.2 ml/hour total flow rate. For concentration quantification, heparin was selected as a model biomolecule for HS due to its structural and charge similarities. Heparin was conjugated to Alexa Fluor 488 dye, according to previously described methods http://fiji.sc), then the intensities had background subtracted and were normalized to the minimum and maximum fluorescence values.MBAs http://www.lonza.com) at passage 2. Alternatively, bone marrow mononuclear cells were obtained from Lonza (2M‐125C), and then hMSCs isolated by plastic adherence and culture according to standard methods. This donor  was characterized for its immunophenotype  and multi‐lineage differentiation potential . A third donor  was isolated in the same manner. Human MSCs were expanded in maintenance medium consisting of low‐glucose Dulbecco's Modified Eagle Medium (DMEM‐LG) , 10% vol/vol fetal calf serum (HyClone), and 4 mM l‐glutamine. Cells were plated at 5 × 103 cells/cm2 in 150 mm culture dishes and grown in a 5% CO2 humidified atmosphere at 37°C. Medium was exchanged every 3 days and cells were passaged at ∼90% confluence. Cells were used at passage numbers as indicated. All media were supplemented with 1% vol/vol penicillin/streptomycin.Normal hMSCs were obtained from Lonza  comprising maintenance medium supplemented with 1 µM dexamethasone, 10 µM insulin, 20 µM indomethacin, and 115 µg/ml 3‐isobutyl‐1‐methylxanthine. Cells were maintained in AIM for 14 days, with AIM prepared fresh and replaced every 3–4 days. Undifferentiated cells used as controls (for each lineage) were kept in maintenance media for 14 days with replacement every 3–4 days.Human MSCs were plated in triplicate at 1.8 × 103 cells/cm2 in 12‐well plates and allowed to attach overnight. Then, medium was replaced with Osteogenic Induction Medium (OIM) comprising of maintenance medium supplemented with 10 nM dexamethasone, 25 µg/ml l‐ascorbate‐2‐phosphate and 10 mM glycerol‐2‐phosphate. Cells were maintained in OIM for 14 days and medium was changed every 3–4 days.Human MSCs were plated in triplicate at 3 × 104 cells) were pelleted in triplicate in Chondrogenic Induction Media  supplemented with 10 ng/ml TGF‐β3 (R&D Systems) in 15 ml centrifugation tubes. Cells were maintained in CIM for 14 days and medium was changed every 3–4 days.Human MSCs (2.5 × 10https://www.bdbiosciences.com) and with FlowJo software v7.6.5 . Gates were set for a false‐positive rate of <2% based on the respective isotype control. For each sample, >10,000 events were acquired. All antibodies were purchased from BD Biosciences, except STRO‐1 .TrypLE‐dissociated hMSC suspensions were stained (as described previously http://www.mn-net.com/) following manufacturer's instructions. Total RNA from chondrogenic differentiated and undifferentiated cells was isolated using TRIzol Reagent following manufacturer's instructions. The quality and quantity of total RNA was determined by a NanoDrop spectrophotometer (Thermo Fisher Scientific). Total RNA (1 μg) was reverse‐transcribed into cDNA using a SuperScript VILO cDNA synthesis kit and an ABI Veriti 96‐well thermal cycler (Life Technologies). Expression levels of target genes were determined using 40 ng of each cDNA sample assayed in triplicate and amplified with a QuantStudio 6 Flex Real‐Time PCR system. The probes for ALPL (liver/bone/kidney isoenzyme), IBSP , PPARG, CEBPA, SOX9, COL2A1, ACAN, and reference gene ACTB (beta‐actin) were purchased pre‐designed . Expression levels were reported in Relative Expression Units (REU) normalized to ACTB.Gene expression was measured in triplicate cultures using reverse transcription and TaqMan hydrolysis probes. Total RNA from adipogenic and osteogenic differentiated and undifferentiated cells was isolated using a Nucleospin RNA extraction kit  situated outside the incubator, driving 1 ml syringes (BD) connected through polyethylene tubing  and stainless steel fittings to the MBA housed in a petri dish inside the incubator.The MBA experimental workflow consists of: MBA preparation and cell seeding and attachment, cell culture, and cell endpoint analysis. MBAs were first autoclaved then vacuum filled with sterile PBS. Human MSCs were detached with TrypLE, washed, and resuspended in fresh medium at 1 × 10l‐glutamine +1% penicillin/streptomycin) with 0.1% bovine serum albumin (BSA) as a carrier protein to prevent adsorption losses in tubing and fittings. Serum was then introduced uniformly in the third factor channel. Final concentrations are as marked.Because serum contains heparin‐binding compounds, the sequential mixing of factors in the MBA was exploited to firstly pre‐complex FGF‐2 with HS8 in the absence of serum. These factors were diluted in minimal medium  and CD90‐PE . Actin and nuclei were optionally counterstained with rhodamine‐phalloidin (1:500) and Hoechst 33342 (2 μg/ml), respectively.At the experimental endpoint, MBAs were removed from the incubator and submerged in a bath of ice‐cold PBS. Fixation and immunostaining were then performed using the syringe pump setup to exchange staining solutions. In each case a minimum volume of 300 μl was delivered to ensure complete fluid replacement within the chip. Antibodies used were: Ki67 , and MetaMorph control and acquisition software . The ‘Scan Slide’ application in MetaMorph was used to acquire the full cell culture array section (∼63 × 23 mm), with a ×4 objective, 2 × 2 camera binning, and in multiple fluorescence channels.Immunostained MBAs were imaged with an Olympus IX83 inverted fluorescence microscope, with an automated stage, CoolSNAP HQ2 CCD camera . Image cytometry processing was performed with CellProfiler software  + nuclei using segmentation based on Ki67 staining. Projected cytoplasmic areas were identified using CD90 staining, with identified nuclei used as the seed structures for shape propagation, and then object measurements were exported.MBA image tiles were stitched into full‐resolution images using MetaMorph, and then these images were registered and sliced into images of individual MBA chambers using a custom MATLAB script  kit (R&D Systems) according to the manufacturer's instructions.Human MSCs were plated in triplicate at 1 x 10https://www.microsoft.com), and color‐scaled with white point at the mean. MINITAB http://www.minitab.com) was used perform factorial analyses. Prism 5  was used for comparisons by analysis of variance (ANOVA) with post hoc tests, as labeled. For factorial analyses, column means  were used, with replicates coming from independent runs from multiple donors. To compare multiple MBA runs, column mean data from each run were standard normalized to arrive at a Z‐score  by: Z=x−μ/σ, where Z is the standard normalized Z‐score for data point x, and μ and σ are the mean and standard deviation of all data points for that run, respectively.Heat maps were generated with Excel , and to detect the cell membrane (CD90 staining) , and the total number of nuclei per chamber  were used as readouts of the cell proliferative and expansion responses to factor treatment. These metrics were arranged as heat maps showing absolute values for each individual chamber in the MBA, versus the combinatorial panel of factors applied to each column in the MBA  between the two metrics, showing chambers with the most proliferating cells (Ki67+ nuclei) also had higher cell numbers after the 3‐day culture period. Distinct hotspots of cell response resulted from treatment with specific combinations of factors  or HS8 (50 µg/ml) resulted in a better coverage of CD90her Fig. A.+ nuclei and total nuclei, suggesting induction of hMSC proliferation and expansion  when comparing variation of the 10 culture chambers in the column  to last chamber (Row 10) in a column, the changes generally presented with smooth gradients    Fig. A. When t 9) Fig. A. Conver 5) Fig. B. Adding 5) Fig. C. Modeli 5) Fig. D.+ was dependent on the downstream position and the response profile was modulated with various treatments of FGF‐2 and HS8 . Overall, the responses of Donors B and C reflected most features of Donor A, providing confidence that factor effects apply to hMSCs generally. However, donor‐to‐donor variability in factor responses was also measured, particularly prominent in Donor C, for which factor sensitivity seemed weaker .To statistically confirm the effects of HS8 and FGF‐2 on hMSC proliferation and expansion, MBA runs were repeated and also completed with additional hMSC donors. Human MSCs (Donors B and C) were derived from bone marrow mononuclear cells by plastic adherence selection and culture, and assayed in a manner similar to Figure + nuclei and total nuclei. Plotting the F‐value from factorial analysis, which measures the relative significance of a factor's effect, showed that significant (p < .05) factor effects were clearly apparent for FGF‐2 and HS8 , meaning there was some donor dependence on the proliferative response to HS8  terms removed by backward elimination, FGF‐2 and HS8 were the only terms left in the model, as well as Donor*HS8, for Ki67+ Nuclei. Plots of fitted means for these individual factor effects for FGF‐2 and HS8 on Ki67+ nuclei and total nuclei all showed positive increasing relationships  from 2 runs from each of the 3 donors A, B, and C were standard normalized and pooled. These data showed the progressively additive effects of FGF‐2 and HS8, until the maximum average responses across the multiple donors and runs were obtained with 50 ng/ml FGF‐2 and 50 μg/ml HS8 , returning to baseline levels after 3 days  were grown for 1 or 3 days in the presence of control medium, and media containing HS8, FGF‐2, or both HS8 and FGF‐2. Then, FGF‐2 levels in the supernatant medium and the cell surface fraction (isolated with a 2 M NaCl wash) were measured by ELISA Fig. . These mays Fig. A. In parays Fig. A and proays Fig. B of FGF‐p > .05 vs. control) and the FGF‐2 in the supernatant decreased  from the level at 1 day (512 pg/well). However, adding HS8 together with FGF‐2 (Both) over a 3‐day period elevated FGF‐2 levels were detected relative to FGF‐2 alone, both on the cell surface  and in the supernatant . It should be noted these data were not normalized to cell numbers, which could account for some differences in total FGF‐2 levels, however the maximum increase in cell numbers observed in the MBA experiments was only to 1.7‐fold of control conditions. Together, these data suggest a dual role for HS8 acting on endogenous and exogenous FGF‐2. HS8 increases the levels and availability of endogenous FGF‐2, and also stabilizes exogenous FGF‐2 over extended periods. Both these mechanisms contribute to a greater integrated amount of FGF‐2 being available to the culture over time.Next, in conditions where exogenous FGF‐2 was supplemented, FGF‐2 could be detected at highly elevated levels both on the cell surface and in the supernatant after 1 day, relative to control cultures. By 3 days, however, the cell surface FGF‐2 had returned to baseline levels  that permits combinatorial assessment of exogenous and paracrine factors, we surveyed hMSC responses to such factor combinations. This improved strategy allowed us to decipher combinatorial effects of ligands like FGF‐2 and the affinity‐purified GAG HS8 on hMSC growth by monitoring direct and paracrine‐dependent cell responses. This approach provides unique insight because the MBA simultaneously provides combinatorial mixes of factors to hMSCs under continuous flow, which allows for the influence and identity of paracrine factors to be probed, using cell readouts along with blocking antibodies, signaling pathway inhibitors, or conditioned medium In the MBA, continual flow of medium is provided in a 100 μm‐high channel directly over the cell layer. Since cells in the first row (chamber) of a column of 10 chambers see fresh medium, and cells in subsequent rows see progressively more depleted and conditioned medium, the row coordinate acts as a surrogate measure of the degree of depletion  and conditioning (accumulation of wastes and secreted endogenous factors) of the medium. Because nutrient provision and waste accumulation are controlled (within a viable range for cell growth) by matching the medium exchange rate to time‐averaged static conditions, we expect most of the variation down a column to be caused by differing levels of exogenous and diffusible paracrine factors. Plotting an appropriate cell response against the row coordinate can therefore provide insight into its dependence on these levels. Using this innovative approach, we uncovered paracrine‐dependent modulation of hMSC proliferative responses, which cannot be easily identified in standard static culture systems. However, the detection of paracrine‐dependent responses allowed us to generate hypotheses that could then be verified in static cultures.Because endogenous, cell surface HS binds endogenous FGF‐2 on export and either promotes autocrine association with FGFR or facilitates juxtacrine transfer between touching cells Exogenous FGF‐2 normally undergoes degradation  can cause disruption of native growth factor‐HSPG feedback loops to alter the distribution of FGF‐2 molecules, thereby enhancing hMSC expansion. Importantly, using an MBA screening strategy, we were able to determine that several regulated systems are modulated by HS8: levels of growth factor production, stability and transport mechanisms. This results in an overall increased level of growth factor cycling through the critical FGF‐HS‐FGFR system, and consequently gains in expansion of the hMSC population.HS8 was shown to act additively with FGF‐2 for enhanced ex vivo expansion of hMSCs. Microbioreactor array experiments and measurements of FGF‐2 levels in static cultures suggest HS8 increases the overall amount and duration of FGF‐2 available to the whole population of hMSCs in culture. This occurs by increasing production and facilitating diffusion of endogenous FGF‐2, and stabilizing exogenous FGF‐2 within the supernatant. Supplementation of hMSC cultures with HS8 could therefore be an effective strategy to enhance hMSC expansion, especially in large‐scale bioreactor systems.D.M.T: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; C.T.: collection and/or assembly of data, manuscript writing, final approval of manuscript; N.G.: provision of study material or patients, final approval of manuscript; V.N.: conception and design, financial support, final approval of manuscript; J.C.‐W.: financial support, provision of study material or patients, final approval of manuscript; S.C.: conception and design, financial support, provision of study material or patients, data analysis and interpretation, manuscript writing, final approval of manuscript.The authors indicate no potential conflicts of interest.Supporting Information.Click here for additional data file.


GBA) causing neuropathic Gaucher's disease (GD) in homozygotes lead to aggressive cognitive decline in heterozygous Parkinson's disease (PD) patients, whereas non‐neuropathic GD mutations confer intermediate progression rates.We hypothesized that specific mutations in the β‐glucocerebrosidase gene  from seven cohorts were analyzed. Differential effects of four types of genetic variation in GBA sequencing carried a mutation. Carriers of neuropathic GD mutations (1.4% of patients) had hazard ratios (HRs) for global cognitive impairment of 3.17  and a hastened decline in Mini–Mental State Exam scores compared to noncarriers (p = 0.0009). Carriers of complex GBA alleles (0.7%) had an HR of 3.22 . By contrast, the common, non‐neuropathic N370S mutation  or nonpathogenic risk variants  did not reach significance.Overall, 10.3% of patients with PD and GBA gene pathogenic for neuropathic GD and complex alleles shift longitudinal cognitive decline in PD into “high gear.” These findings suggest a relationship between specific types of GBA mutations and aggressive cognitive decline and have direct implications for improving the design of clinical trials. Ann Neurol 2016;80:674–685Mutations in the  Initial motor features of Parkinson's disease (PD) typically respond to dopaminergic medications. Dopaminergic therapies do not slow the underlying neurodegenerative disease process. Over time, the neuropathology spreads, and, in many patients, dementia emerges as one of the most debilitating and intractable complications of the disease.GBA) enriched in severe, neuropathic Gaucher's disease (GD), but not those typical of mild, non‐neuropathic GD, will be associated with an aggressive cognitive decline in PD. Two mutant copies of GBA cause GD, the most prevalent lysosomal storage disease. This autosomal‐recessive disorder is linked to over 300 pathogenic mutations in the GBA gene,We hypothesized that mutations in the glucocerebrosidase gene  it is important to have the ability to predict the disease course of individual patients. It is thus of practical importance to precisely understand the relation between specific types of GBA mutations and the speed of PD progression. Here, we determined that GBA mutations linked to neuropathic GD, but not those of non‐neuropathic GD, are associated with a more rapid longitudinal cognitive decline in seven international cohorts representing 2,304 patients with PD longitudinally evaluated for up to 12.8 years  with 20,868 study visits.Individuals carrying one mutant copy of LRRK2 G2019S mutation were excluded.Seven longitudinal cohortsGBA coding sequence and flanking intronic regions in four data sets . To avoid sequencing its neighboring pseudogene, the entire locus was amplified in a single long‐range polymerase chain reaction (PCR) reaction using the LA PCR Kit . Template DNA (100ng) was added to a 50‐µl reaction along with primers  with the following sequences: forward primer (5′‐CGACTTTACAAACCTCCCTG‐3′) and reverse primer (5′‐CCAGATCCTATCTGTGCTGG‐3′), and cycling conditions were 94°C for 1 minute (one cycle), 98°C for 10 seconds followed by 68°C for 15 minutes (30 cycles), and 72°C for 10 minutes (one cycle). This long‐range PCR assay uses primers that target sequences that uniquely flank GBA and produces a single 7,755‐base‐pair (bp) PCR product. PCR products were visualized on an 0.8% agarose gel with ethidium bromide to confirm successful amplification, which were then used to construct Illumina ready sequencing libraries using the NexteraXT kit , following the manufacturer's instructions. Uniquely indexed samples were pooled (up to 384 samples/pool) and run on the Illumina MiSeq instrument to generate 150‐bp paired‐end reads. Sequencing reads were aligned to the human assembly genome (GRCh37/hg19) using BWAGBA mutations were called by the GATKGBA mutation locus for all replicates. For 383 PD sample in HBS, E326K, N370S, and T369M were previously genotyped.GBA after two‐stage PCR, as part of a previous study.GBA were sequenced. To avoid amplifying and sequencing the neighboring pseudogene, GBA was amplified in three large fragments , using previously described primers and a unique 648C to 548C touch‐down PCR program.GBA coverage was performed. On average, 99.5% of the exonic GBA regions were covered for at least 15×. For the remaining samples, the overall targeted exome coverage (10×) is 90.8%.Mutations were identified through targeted next‐generation sequencing of the entire ts Table . For 173GBA , with Student t tests, and distribution‐free Mann–Whitney U or Fisher's exact tests, as appropriate.Differences in continuous and categorical baseline characteristics were compared between noncarriers and carriers of genetic variation in GBA mutations on cognitive progression in PD. To characterize how distinct types of GBA mutations are associated with longitudinal disease progression in PD, we compared four operationally defined groups of PD patients with distinct types of GBA mutations to patients with PD not carrying a GBA mutation. Patients with PD carrying one of four types of GBA mutations or variant were considered and compared to patients with PD not carrying a GBA mutation (noncarriers), respectively. 1, Carriers of a GBA risk variant. The E326K, T369M, and E388K variants are associated with risk of PDGBA mutations, but it is controversial whether they are per se pathogenic for GD.2, Carriers of the common, non‐neuropathic N370S mutation. The pathogenic N370S mutation is classically associated with mild, non‐neuropathic phenotypes of GD type 1. 3, Carriers of a neuropathic GD mutation. For the purpose of this study, patients with PD who were heterozygous carriers of a pathogenic GBA mutation associated with neuropathic GD subtypes 2 or 3 in one or more published reports (found on PubMed searches in September 2015) were operationally defined as “carriers of a neuropathic GD mutation” (Supplementary Table 1). This included carriers of the L444P mutation, the prototypical mutation associated with neuropathic GD types 2 and 3. 4, Carriers of complex GBA alleles. Patients carrying complex GBA alleles  were grouped separately. We also explored longitudinal cognitive decline in patients with PD carrying any pathogenic GBA mutation or risk variant taken together  compared to patients with PD not carrying a GBA mutation.The primary aim of this study was to analyze the effect of four types of p values were obtained by penalized quasi‐likelihood ratio tests of the full model with the effect in question contrasted with the model without the effect in question. These analyses were implemented in Rp values less than or equal to 0.05 were considered statistically significant.Generalized longitudinal mixed fixed‐ and random‐effects analysis of cognitive decline was performedCox proportional hazards models were used to estimate the influence of carrier status on the hazard ratio of time to the endpoint reaching global cognitive impairment as indicated by a MMSE ≤25 according to the recommendation the International Parkinson and Movement Disorder Society (MDS) Task Force.GBA mutation, we ran a power analysis. We used a repeated‐measures analysis of variance design of two groups versus four time points . One hypothetical group was assigned to placebo and therefore stipulated to have MMSE means across time predicted by our mixed‐effects model, and the second group was assigned to treatment with an experimental drug, which has the hypothetical ability to halt decline in MMSE scores . We ran analyses assuming a two‐tailed α of 0.05 to detect the difference in trajectories across time for the two groups (group × time interaction), and assuming a within group/time‐point standard deviation (SD) of 2 and a 1‐year test‐retest correlation of 0.7 as approximate to those values found empirically, with a first‐order autoregressive decay across longer periods. The conservative Greenhouse‐Geisser correction for degrees of freedom for correlated error was also used. For comparison, analogous computations were performed for a hypothetical clinical trial scenario with “all comer” PD patients (not carrying a GBA mutation).To estimate sample‐size requirements for a personalized, 3‐year clinical trial of a hypothetical drug designed to halt cognitive decline (as measured by the MMSE) specifically in patients carrying a neuropathic GBA mutation) were enrolled to test the same experimental drug , 893 patients would be required per placebo and 893 per drug group to achieve 80% power R, and R463P) were detected. The R463P and K(‐27)R mutations have not been conclusively associated with a GD subtype and were thus excluded from the mutation‐type specific analyses. D140H mutations identified were part of complex alleles  compared to noncarriers (p = 0.0009), adjusting for age of onset, sex, years of education, and study  compared to noncarriers (p = 0.022), adjusting for age of onset, sex, years of education, and study  compared to noncarriers, adjusting for age of onset, sex, years of education, and study  compared to noncarriers, adjusting for age of onset, sex, years of education, and study  compared to noncarriers (p = 0.0005), adjusting for age of onset, sex, years of education, and study .We then explored the effect of GBA mutations enriched in neuropathic GD in homozygotes  were associated with aggressive cognitive decline in PD. Patients carrying these mutations had an HR of 3.17  for developing global cognitive impairment compared to patients without a mutation — a 217% risk increase. By contrast, patients carrying the common N370S mutation linked to non‐neuropathic GD showed a trend toward intermediate rates of longitudinal cognitive decline. This did not reach statistical significance, likely due to insufficient power. This report is the first to evaluate the differential effects of distinct types of neuropathic, complex alleles, non‐neuropathic, and risk variation in GBA on the longitudinal trajectory of cognitive decline in PD. It suggests the clinical‐genetic concept of aggressive, neuropathic GBA‐PD. The cross‐sectional observation that PD is more frequent among carriers of a neuropathic GD mutations, compared to carriers of non‐neuropathic GD mutations in an Ashkenazi‐Jewish population,Seven deeply phenotyped, longitudinal cohorts from North America and Europe representing 2,304 patients with PD were followed longitudinally for up to 12.8 years  with a total of 20,868 in‐person study visits. This makes the current analysis one of the largest longitudinal observational studies reported for PD. Much progress has been made in delineating genome variation associated with susceptibility for developing PD,GBA‐PD into genetics‐guided, proof‐of‐concept trials for disease‐modifying precision therapeutics may increase power, shorten trial duration, shrink sample sizes, and, possibly, cut costs. Our hypothetical power analysis estimates that a trial targeted to neuropathic GBA‐PD could shrink the sample size required by as much as 25‐fold compared to an equally powered trial of PD patients without a GBA mutation  enzymatic activity.GBA loss‐of‐function and toxic gain‐of‐function hypotheses have been proposed.GBA promoted α‐synuclein accumulation in a dose‐dependent manner without observed loss of GCase function.GBA mutation, GCase activity is reduced in induced pluripotent stem‐cell–derived neurons,GBA mutation, although to a lesser extent than those with a mutation.GBA‐PD could also be of benefit for PD patients without a GBA mutation.The mechanism through which In GD, enzyme replacement therapy is effective in reversing peripheral disease manifestations, but does not penetrate the blood–brain barrier. In an encouraging development for both neuropathic GD and PD, small molecules designed to correct the GCase pathway in the brain are in development.G.L., B.B., J.J.L., B.R., J.J.v.H., J.‐C.C., R.A.B., J.M., C.H.W.‐G., and C.R.S. were responsible for conception and design of the study. All authors were responsible for acquisition and analysis of data. G.L., B.B., J.J.L., R.A.B., C.H.W.‐G., and C.R.S. were responsible for drafting a significant portion of the manuscript or figures.C.R.S. has a scientific collaboration with Genzyme.Additional supporting information can be found in the online version of this articleSupporting InformationClick here for additional data file.