A novel approach for the isolation and long-term expansion of pure satellite cells based on ice-cold treatment

Anna Benedetti
Gianluca Cera
Daniele De Meo
Ciro Villani
Marina Bouche
†
Biliana Lozanoska-Ochser
A novel approach for the isolation and long-term expansion of pure satellite cells based on ice-cold treatment
10.1186/s13395-021-00261-wM E T H O D O L O G Y Open AccessSatellite cell isolationSatellite cells in vitro expansionSkeletal muscle regeneration
Satellite cells (SCs) are muscle stem cells capable of regenerating injured muscle. The study of their functional potential depends on the availability of methods for the isolation and expansion of pure SCs with preserved myogenic properties after serial passages in vitro. Here, we describe the ice-cold treatment (ICT) method, which is a simple, economical, and efficient method for the isolation and in vitro expansion of highly pure mouse and human SCs. It involves a brief (15-30 min) incubation on ice (0°C) of a dish containing a heterogeneous mix of adherent muscle mononuclear cells, which leads to the detachment of only the SCs, and gives rise to cultures of superior purity compared to other commonly used isolation methods. The ICT method doubles up as a gentle passaging technique, allowing SC expansion over extended periods of time without compromising their proliferation and differentiation potential. Moreover, SCs isolated and expanded using the ICT method are capable of regenerating injured muscle in vivo. The ICT method involves minimal cell manipulation, does not require any expertise or expensive reagents, it is fast, and highly reproducible, and greatly reduces the number of animals or human biopsies required in order to obtain sufficient number of SCs. The cost-effectiveness, accessibility, and technical simplicity of this method, as well as its remarkable efficiency, will no doubt accelerate SC basic and translational research bringing their therapeutic use closer to the clinic.

Background

The muscle is endowed with an exceptional regenerative ability primarily due to a resident population of stem cells called satellite cells (SCs) [1][2][3]. Ensconced between the basal lamina and the plasma membrane of muscle fibers, SCs respond to injury or various stress stimuli by becoming activated, and undergoing proliferation, selfrenewal, and differentiation to form new myofibers [4,5]. As SCs become activated, a proportion of them identified as Pax7 + MyoDreplenish the stem cell pool, while others acquire the expression of MyoD (Pax7 + MyoD + ), differentiate into myoblasts and enter the myogenic program. After several rounds of division myoblasts give rise to Myogenin + myocytes, which fuse together to form new myofibers [1,4,[6][7][8][9].

A major stumbling block in the study of the functional potential of SCs has been the lack of isolation methods involving minimal cell manipulation and allowing the isolation and expansion of highly pure SCs with preserved myogenic properties after serial passages in vitro [10][11][12]. Moreover, the success of SC transplantation therapy depends on having an efficient method to isolate and expand these cells in vitro in undifferentiated state and in sufficient numbers [13].

Presently, there are three main methods commonly used for the isolation of SCs: the pre-plating method, fluorescence activated cell sorting (FACS), and magnetic bead isolation method.

The pre-plating method is based on the differing adhesive properties of muscle cells, with SCs being the least adherent. Following enzymatic digestion, a heterogeneous mix of skeletal muscle cells is plated onto uncoated culture dishes and after a 1-24-h incubation at 37 ○ C, the non-adherent cells are collected and plated onto new collagen coated dishes [10][11][12]14]. The resulting cell culture contains both SCs and fibroblasts in variable proportion. To improve SC purity, the pre-plating step can be repeated every 24 h over 6 days [10]. Although cheap and straightforward to perform, this method's main disadvantage is that it is time consuming and gives rise to cultures of variable purity, with fibroblast contamination and overgrowth by day 7 of culture, leading to early senescence and detachment of myotubes [10]. A recently described version of the pre-plating method introduces a re-plating step whereby after a 2-day expansion, the adhered cells are detached with trypsin and replated onto Matrigel coated dishes, giving rise to SC cultures of much improved purity [15].

The FACS sorting method sorts muscle mononuclear cells pre-labelled with SC specific antibodies. Following digestion of muscle with various enzymes the resulting mixture of cells is labelled with specific antibodies to facilitate the identification of SCs, which are then sorted using a FACS sorter instrument [16][17][18][19][20][21]. At present, the FACS sorting method represents the gold standard for the isolation and study of SCs. Nevertheless, there are several disadvantages to this method including high cost and the requirement for a FACS sorter instrument. Moreover, this method is time consuming, requires expertise to perform and cell purity can be variable. The cell labelling step followed by the sorting procedure can potentially stress or damage the cells, decrease their viability, or alter their functional properties in vitro [12]. The third method is based on magnetic cell separation (MACS) and uses magnetic columns and SC specific magnetic bead kits [22]. It is based on negative selection of SCs by magnetically labelling and removing other cell lineages. Because this method assumes that all the other cell types are successfully removed from the muscle cell preparation, it is less precise than the FACS sorting method. This method is expensive to perform, time consuming, and stressful for the cells. As for the other two methods, cell purity is variable and often the SC cultures become overgrown by fibroblasts by day 7 [10,12].

There is therefore a need for new and improved methods for the isolation, expansion and culture of SCs. Here, we describe a simple, inexpensive, and efficient method for the isolation of highly pure mouse and human SCs that can be serially expanded in vitro to obtain sufficient number of SCs with preserved proliferation potential, capable of regenerating injured muscle in vivo.

Methods

Mice C57BL/10ScSn-Dmd mdx , C57BL/10ScSn, and C57BL/6J mice were purchased from the Jackson laboratory (Bar Harbor, ME, USA). Both male and female mice were used. The mice were housed in the Histology Department-accredited animal facility at the University of Sapienza. All the procedures were approved by the Italian Ministry for Health and were conducted according to the EU regulations and the Italian Law on Animal Research.

Human muscle sourcing

Muscle biopsies (gluteus maximus) were obtained from patients (8 males and 7 females, age range 50-90 years) undergoing surgery at the Department of Orthopaedics and Traumatology, Umberto I Hospital in Rome, Italy. According to the Italian law, the authors are not required to ask for approval from an institutional review board or ethical committee for this type of study. In any case, all patients gave their approval to undergo intraoperative muscle biopsy and to publish the clinical and laboratory data obtained.

Satellite cell isolation with the ice-cold treatment method

SCs were isolated from hind-limb muscles of 4-8-weekold mice or from human biopsies. Muscles were dissected with scissors and finely diced with a scalpel in a dish containing DMEM (Sigma-Aldrich, St. Louis, MO, USA, D5671). This was followed by enzymatic digestion with 10 ml/g of muscle of Collagenase type II (Sigma-Aldrich, SCR103) at a concentration of 0.4 mg/ml in PBS (Sigma-Aldrich), for 45 min in a water bath at 37°C with agitation. Digestion was blocked with DMEM 10% FBS and after centrifuging the muscle preparation and removing the supernatant, a second digestion was performed with 10 ml/g of muscle of Collagenase/Dispase at a concentration of 1 mg/ml (Roche, Basel, CH, 11097113001) in PBS Calcium-Magnesium free (Sigma-Aldrich), for 30 min at 37°C in a water bath with agitation. The digested muscle was then passed first through a 70-μm cell strainer followed by 40-μm cell strainer to obtain single cell suspension. Next, the cells were centrifuged, resuspended in DMEM 10% FBS (Sigma-Aldrich, F2442), counted and plated at 2 × 10 6 cells/100 mm dish (uncoated) (Corning, NY, USA, 430167), and incubated at 37°C for 1 h. Non-adherent cells were collected, centrifuged, and the cell pellet was resuspended in DMEM 10% FBS, plated again, and incubated for another 1 h at 37°C. After the second pre-plating, non-adherent cells were collected, centrifuged, counted, resuspended in SC Growth Medium (GM) DMEM, 20% Horse Serum (Thermo Fisher Scientific, Waltham, MA, USA, 26050088), 3% Chicken Embryo Extract (Seralab, CE-650-J), and plated into 100 mm dishes coated with 0.1% gelatin (Stem Cell Technologies, Vancouver, BC, CAN, 07903), at 10 6 cells/dish. The next day, the dishes containing a heterogeneous mix of adhered muscle cells were washed 3 times with PBS, and 10 ml of ice-cold PBS was added into each dish. The dishes were then placed on ice (0°C) for 15-30 min with occasional gentle manual shaking (swirling motion). The detached cells were collected, centrifuged, resuspended in GM, and plated into 0.1% gelatin-coated 35-mm dishes (Corning, 353001) at a density of 10 3 cells/dish. To induce differentiation, proliferating cells (day 3 after adding GM) were cultured in differentiating medium (DM) containing DMEM 5% Horse Serum, 1% Chicken Embryo Extract.

Satellite cell isolation with magnetic bead labelling

Mouse SC isolation by magnetic bead labelling was performed by using a SC Isolation Kit (Miltenyi Biotech, Bergisch Gladbach, DE, REF: 130-104-268) as previously described [23]. Briefly, minced muscle was digested as described above. The digested muscle was passed through 70 μm and 40 μm cell strainers, and the resulting single cell suspension was centrifuged, resuspended in 80 μl buffer (PBS pH 7.2, 0.5% FBS, 2 mM EDTA), and incubated with 20 μl of Satellite Cell Isolation Kit per gram of muscle, for 15 min at 4°C. Next, the cell suspension was passed through a LS column (Miltenyi Biotech, 130-042-401) placed in a magnetic field of a MACS Separator (Miltenyi Biotech). Unlabeled SCs were collected in the flow-through, counted, washed, resuspended in growth medium (GM), and plated into 35mm dishes at a density of 10 3 cells/dish.

SC detachment with trypsin

SCs were rinsed once with PBS and then incubated with trypsin-EDTA solution (Sigma-Aldrich, T3924) for 5 min at 37°C. SCs were then collected, centrifuged, resuspended in GM, and plated at a density of 10 3 cells/ dish.

SC transplantation

Acute muscle injury was induced the day before SC transplantation. To induce muscle injury tibialis muscles were injected with 0.01 ml of Cardiotoxin from Naja Pallida (10 μM) (Latoxan ZA, Les Auréats, Fr), using a 30 Gauge micro-syringe [23][24][25][26].

For cell transplantation, 15,000 SCs were resuspended in 20 μl of DMEM 2% FBS (Sigma-Aldrich) and injected into the TA muscle of one leg with a single injection by using a 30 Gauge micro-syringe. Contralateral TA muscle was injected with only PBS and used as control.

Immunofluorescence and microscopy

For immunofluorescence analysis cultured SCs were fixed in PFA 4% for 10 min RT, permeabilized in cold methanol at -20°C for 6 min, blocked in 5% Goat Serum in PBS for 30  For immunofluorescence analysis of mdx TA muscle transplanted with WT SCs, 8-μm-thick muscle cryosections were fixed in 4% PFA for 10 min at room temperature (RT), and then permeabilized in cold methanol for 6 min at -20°C. Sections were then blocked in 5% Goat Serum (Sigma-Aldrich) in PBS for 30 min at RT. Next, sections were incubated with primary rabbit anti-dystrophin antibody (1:200, Abcam, Cambridge, UK) overnight at 4°C. The next day, sections were washed and incubated with a secondary antibody goat anti rabbit Alexa Fluor 488 (1:1000 Abcam). Nuclei were counterstained with Hoechst.

Bright field images were acquired with an inverted phase-contrast microscope (Nikon Eclipse, TS100). Images were acquired with a Nikon DS-Fi2 camera and NIS Elements version 4.0 Imaging System.

CFSE staining

Isolated SCs were stained with CFSE (ThermoFisher Scientific) at a concentration of 1 μM for 10 min at 37°C in the dark prior to culture. After 4 days of culture, the cells were detached with Accutase Solution (Sigma-Aldrich). Samples were acquired with a CyAn ADP (DAKO) flow cytometer and acquired data were analyzed using FlowJo software version 10 (FlowJo LLC, Ashland, OR, USA).

RNA isolation and quantitative real-time PCR

For RNA preparation, cells were lyzed with TRI reagent (Sigma-Aldrich) and processed as previously described [23]. Reverse transcription was performed with Sensi-FAST™ cDNA Synthesis Kit (Bioline, Memphis, TN, USA). Quantitative real-time PCR assays were performed according to the MIQE criteria, using Sensi-FAST™ SYBR No-ROX Kit (Bioline) following manufacturer's protocol. All reactions were performed in duplicate. Data were collected and analyzed using ABI PRISM 7500 Sequence Detection System (Life Technologies, Carlsbad, CA, USA). Quantitative RT-PCR values were normalized to the expression of GAPDH mRNA. The relative gene expression level was calculated using the 2−ΔΔCT method and reported as mean fold change in gene expression.

Clonal myogenicity assay

For the clonal myogenicity assay, SCs were plated into 0.1% gelatin coated 96-well plates, (excluding the outer wells of the plate) at 1 cell per well, in growth medium. Colony formation and number of cells were assessed at 24, 48, and 72 h of culture.

Statistical analysis

All statistical analyses were performed using Graph-Pad Prism software version 8 (La Jolla, CA, USA). Data are presented as mean ± SEM. Statistical significance was determined using unpaired 2-tailed Student's t test with Welch's correction for unequal variances. A P value of ≤ 0.05 was considered statistically significant.

Results

Isolation and characterisation of muscle SCs using the icecold treatment method

Previous studies have demonstrated that cold temperature causes a reduction in cell adhesion, likely due to the downregulation of adhesion receptors [27,28]. Compared to other cells, such as fibroblasts, which typically contaminate SC cultures, SC are considered to be less adherent [11,14]. Taking this into consideration, as well as the notion that like all stem cells, SCs are sensitive to stress signals and are among the first muscle resident cells to respond to injury [4,5], we hypothesized that subjecting a heterogeneous culture of muscle cells to a mild stress stimulus such as ice-cold temperature will lead to the detachment of only the SCs. To test this hypothesis, we obtained a mix of muscle cells following enzymatic digestion, and after 2 h of preplating on uncoated dishes, cultured them overnight on gelatin-coated dishes. The next day, after washing and removing the non-adhered cells and debris, we placed the dishes of heterogeneous muscle cells on ice for 30' (Fig. 1a, b). This time point was chosen based on the purity and number of SCs obtained following 15, 30, 45, and 60' on ice. Although the number of SCs obtained increased with longer incubations, the purity of SCs decreased from 100% at 15-30' to 95 and 90% at 45 and 60' on ice, respectively (data not shown). The ice-cold treatment (ICT) method can be used to harvest SCs from the original dish containing the heterogeneous muscle cells for at least the first 3 days of culture. Placing the heterogeneous muscle culture dish on ice for 30' resulted in the detachment of only the SCs giving rise to a highly pure culture of SCs that proliferated and differentiated into myotubes upon culture in differentiating medium (Fig. 1b-d). Satellite cells isolated using the ICT method were 100% pure as determined by the expression of the SC markers Pax7 and MyoD (Fig. 1c). At day 3 of proliferation, 100% of the cells were positive for the satellite cell marker Pax7 and of these 97% were activated and expressed MyoD (Pax7 + MyoD + ) (Fig. 1c). To examine the myogenic capacity of ICT-isolated SCs we performed a clonal myogenicity assay, by plating a single cell per well and analyzing the formation of myogenic colonies. Satellite cells isolated with the ICT method displayed a similar clonal myogenicity of 40 % and a doubling time of 17 h to SCs isolated using the magnetic beads method (Figure S1A and B). The myogenic identity of the cultured cells was further confirmed by the expression of myosin heavy chain (MHC) and the formation of myotubes. After 3 days in differentiating medium the SCs differentiated into myoblasts that fused into MHC expressing myotubes with a fusion index of 90% (Fig. 1d). The purity of the isolated SCs at the beginning of culture, as well as the fusion index and number of myonuclei per myotubes were similar between SCs isolated with the ICT and magnetic cell separation (MACS) method ( Figure S1C-E). However, by day 5-7, the cultures obtained with the MACS method became overgrown by non-myogenic cells such as PDGFRα + fibroblasts, causing premature myotube detachment, whereas cultures obtained with the ICT method remained almost free of contaminating cells (Fig. 1e, f). Overall, these results confirm our hypothesis that ICT leads to the preferential detachment of SCs and using this method allows the isolation of 99-100% pure SCs.

The ICT approach can also be used for serial passaging and long-term expansion of SCs A major obstacle in SC research has been that cultured SCs lose their proliferation potential after a couple of passages and begin to differentiate into myotubes thereby limiting the number of SCs that can be serially expanded in vitro. Loss of differentiation potential has also been noted with increasing number of passages [10][11][12][13]29]. We reasoned that, since the ICT leads to SC detachment, it can also be used to passage growing cultures of proliferating SCs. Indeed, placing the dishes of proliferating SCs on ice for 30 min led to the detachment of around 30% of the proliferating cells and we were able to serially passage and expand the proliferating SCs without compromising their proliferation and differentiation capacity (Fig. 2a). We used the ICT approach on proliferating SCs and then successively on each established culture of proliferating SCs (for more than 10 passages). This approach yielded on average 2.5 × 10 6 cells/g of muscle (Fig. 2b). The SCs passaged using the ICT method did not lose their proliferation and differentiation potential and displayed a minimally altered Pax7 and myogenin gene expression (Fig. 2c, d). While ICTpassaged SCs retained their proliferation and differentiation capacity even after 10 passages, SCs passaged with the most commonly used passaging reagent trypsin, lost the ability to form new myogenic colonies after just 2 passages and instead begun to differentiate into myotubes ( Fig. 2e-g). Similar to SCs detached by trypsin, SCs detached using a gentler detachment solution such as Accutase, exhibited accelerated differentiation after more than 3 passages (data not shown).

Previous studies have demonstrated that based on their rate of proliferation, SCs can be divided into two subpopulations: fast dividing and slow dividing [30]. The slow-dividing are a subset of SC that have been shown to retain stemness and long-term self-renewal ability [31]. Unlike the fast-dividing SCs, which have a limited ability to form secondary myogenic colonies after passage and instead undergo differentiation, the slow dividing SCs form secondary myogenic colonies when passaged [30,31]. To examine the prevalence of fast and slow dividing SCs within our population, we labelled the ICT-passaged SCs with CFSE prior to plating and analyzed the rate of proliferation 3 days later. We compared the proliferation rate of ICT-isolated and passaged SCs with that of SCs isolated using magnetic beads and passaged with trypsin. In line with previous observations, we found that most of the activated SCs isolated using the commercial kit and passaged with trypsin were fastdividing cells (CFSE lo ), with slow-dividing cells (CFSE hi ) representing less than 10% of the total. Interestingly, while similarly heterogeneous, the SCs isolated and passaged using the ICT method were enriched in the slow dividing SC population (Fig. 2h, i).

Next, we examined the longevity of differentiated ICTisolated SCs in culture. The highly pure cultures of SCs isolated using the ICT method gave rise to myotubes that could be maintained in culture for up to 1 month, compared to just 7 days when isolated using the magnetic beads ( Figure S2A-C) or the pre-plating method ( Figure S2D).

Overall, these data show that the ICT method can be used for the serial expansion of SCs with preserved proliferation and myogenic potential over an extended period of time compared to other methods.

To examine the potential of ICT-isolated and expanded SCs to regenerate injured muscle, we injected 15,000 SCs isolated from wild-type mice into the tibialis anterior of mdx mice (lacking dystrophin) CTX injured 24 h previously. SCs were either injected immediately after ICT isolation, or after 3-day in vitro expansion following ICT. Transplantation of both SCs immediately after ICT-isolation and after in vitro expansion contributed to the regeneration process to a similar extent as evidenced by the appearance of newly formed dystrophin-positive fibers ( Figure S3A and B). There was no difference in the ability of transplanted ICT-isolated (See figure on previous page.) Fig. 1 Isolation and characterization of muscle SCs using the ICT method. a Schematic representation of the ICT method. b Representative bright field images of the heterogeneous muscle mononuclear cell culture from which SCs were isolated by ICT, and representative images of the ICTisolated SCs at D2 and D4 in growth medium (GM) and at D3 after adding differentiation medium (DM) (n = 15 independent experiments). c Representative immunofluorescence images of ICT-isolated SCs stained for Pax7 (red), MyoD (green), and nuclei (blue) and a graph showing the percentage of cells positive for Pax7 and/or MyoD at day 3 of culture in GM (n = 3 independent experiments). d Representative immunofluorescence images of ICT-isolated SCs stained for myosin heavy chain (MHC) (red) and nuclei (blue) and a graph showing percent fusion after differentiation (4 days in GM followed by 3 days in differentiating medium (DM)) (n = 3 independent experiments). e Representative immunofluorescence images of ICT-isolated SCs and MB-isolated SCs stained for Myogenin (red) PDGFR⍺ (green) and nuclei (blue) and a graph showing percent PDGFR⍺ + cells at day 5 of culture in GM. f Representative bright field images of ICT-and magnetic beads (MB)-isolated SCs and a quantification graph showing percentage of myogenic cells in ICT-and MB-isolated SCs after differentiation at day 7 of culture (n = 3 independent experiments). Non-myogenic cells were identified as Pax7 -MyoDnuclei outside the myotubes. Scale bar = 100 μm. Error bars represent mean ± sem. *p < 0.05 by Student's t test. SCs and SCs isolated by magnetic beads to regenerate injured muscle ( Figure S3C and D). These experiments confirmed that SCs isolated with the ICT method successfully engraft after transplantation and do not lose their potential to regenerate injured muscle after expansion in vitro.

Efficient isolation, serial expansion and long-term culture of human satellite cells with the ICT method

The study of human SCs (hSCs) has generally lagged behind that of mouse SCs due to the difficulties associated with obtaining muscle tissue, as well as the lack of methods for the isolation of pure hSCs that can be expanded in vitro without altering their myogenic potential [13]. Having demonstrated the remarkable efficiency of the ICT method in the isolation of pure mouse SCs, we set to reproduce these findings using human muscle biopsies. We obtained gluteus maximus specimens from patients undergoing surgery, aged between 50 and 90 years. Using the same approach as described above, we consistently and reproducibly obtained a highly pure culture of hSCs (Fig. 3a, d-g) from a heterogeneous population of human muscle cells ( Figure S4), that could be serially expanded for more than 10 passages (Fig. 3a). As previously reported [32], we found that hSCs proliferated slower than mouse SCs, with a doubling time of 46 h (Fig. 3b), reaching a peak around day 10 post isolation, and slowing down thereafter. On average we isolated around 20 × 10 3 SCs/g of muscle. Using the ICT method, these hSCs could be expanded 300-fold over a period of 2 months to a final total of 6 × 10 6 /g of muscle (Fig. 3c). Previous studies have shown that hSCs rapidly downregulate Pax7 expression in culture [33]. In agreement with others [33], we found that the expression of Pax7 was variable and ranged between 45 and 50% at day 2 of culture in GM (Fig. 3d). Almost 100% of the hSCs were myogenin positive at day 5 of culture in GM, suggesting that most of them have activated their myogenic program (Fig. 3e). The myogenic purity of the hSC culture was further confirmed by desmin at day 5 (Fig.  3f) and MHC expression at day 10 after shifting to differentiating medium (Fig. 3g).

These data show that the ICT method performs equally well when used for the isolation of human SCs.

Discussion

Over the past decade, a considerable progress has been made in the development of new methodologies for the isolation of SCs. Nevertheless, each of the available methods suffers from at least one disadvantage be it purity, cost, expertise, or a combination of these [11]. In this study, we describe a novel method for the isolation of pure mouse and human SCs, that is inexpensive, simple to perform, and reproducibly efficient. The ICT method takes advantage of the differing adhesive properties of muscle cells as well as the ability of SCs to rapidly respond to stress stimuli [5,11,14]. Thus, the combination of a mild cold-stress stimulus and cold-induced reduction in adhesion, leads to the detachment of only the SCs. Exposure of mammalian cells to cold stress can slow down the progression through the cell cycle and inhibit protein synthesis. Moreover, depending on the intensity and duration, cold stress can activate the apoptotic program, or lead to necrosis [34]. Thus, it is conceivable that prolonged exposure to cold temperature could interfere with the myogenic properties of SCs. Nevertheless, we found that the brief period of exposure to ice-cold temperature did not interfere with the ability of SCs to proliferate and differentiate in vitro or with their in vivo regeneration potential, suggesting that ICT does not alter SC function. Indeed, SCs isolated by ICT behaved similarly to those isolated using the pre-plating method or the magnetic bead isolation kit. Of note, both the magnetic bead and the FACS sorting isolation methods involve far lengthier incubation times on ice compared to our method without interfering with SC behavior and function in vitro and in vivo [16,17,21,22]. Interestingly, Marg A et al. recently found that storing human muscle biopsies at 4°C, in low serum medium and no O 2 for up to 35 days and subsequent culture at 37°C and 21% oxygen led to SCs expansion outside the fiber fragments. Surprisingly, the purity of the outgrowing colonies of SCs was 100% myogenic cells, since non-myogenic cells such as fibroblasts (See figure on previous page.) Fig. 2 Serial expansion and long-term proliferation potential of SCs using the ICT method. a Representative bright field images of SCs at day 3 of culture in GM following ICT 1, ICT 2, and ICT 3, and differentiated cells at day 3 of culture in DM following ICT 3 (n = 10 independent experiments). b Total number of SCs at day 3 of culture in GM, after 1 and 10 ICTs (n = 10 independent experiments). c Pax7 gene expression in SCs at day 2 of culture in GM after ICT 1 and ICT 2 analyzed by quantitative real time PCR (n = 3 independent experiments). d Myogenin expression in SCs at day 5 of culture in GM after ICT1 and ICT 2 analyzed by quantitative real-time PCR (n = 3 independent experiments). e Representative images of SCs at day 2 of culture in GM, after 1-3 detachments with ICT (top panels) and trypsin (bottom panels). f Pax7 gene expression in SCs detached with ICT or trypsin, at day 2 of culture in GM, analyzed by quantitative real-time PCR (n = 3 independent experiments). g Myogenin gene expression in SCs detached with ICT or trypsin, at day 5 of culture in GM, analyzed by quantitative real time PCR (n = 3 independent experiments). h Representative overlay of histogram plots of CFSE labeled ICT and trypsin detached SCs at time 0 and day 4 of culture in GM. i Graph showing percent CFSE-low or slow-proliferating SCs after ICT or trypsin detachment, at day 4 of culture in GM (n = 3 independent experiments). Error bars represent mean ± sem, *p < 0.05, calculated by Student's t test. did not survive prolonged storage at hypothermic conditions [33]. Therefore, it is likely that SCs tolerance for cold stress is high compared to other cell types, and this trait can be exploited as we did with our ICT method, to improve the purity of SCs grown in vitro.

Recently, Yoshioka K et al. described an improved version of the pre-plating method, reducing the isolation and purification procedure to 2.5 days in total, while increasing the cell yield, and significantly improving the purity of the resulting SC culture by introducing a replating step [15]. The re-plating step performed at day 2.5 of culture involves the detachment of all adhered cells including fibroblasts and SCs with trypsin, and replating on matrigel coated dishes [15]. While the purity of the resultant SC culture is comparable to ours, our method involves fewer steps in total and only one overnight pre-plating. In addition, the ICT method doubles up as a very gentle passaging technique, allowing longterm serial expansion of SCs ex vivo, without altering their proliferation and differentiation properties. Whereas the FACS sorting method is and will remain the gold standard for the study of SCs immediately after isolation, the ICT approach will likely become the method of choice for the in vitro expansion of SCs. After each ICT passage, the already expanded SCs can be cryopreserved, and stored until needed. With the ICT method we were able to passage proliferating mouse and human SCs for at least 10 times, expanding their number 150-and 300-fold, respectively. This represents a clear advantage over the most commonly used passaging reagent trypsin, which we and others have shown, typically accelerates the differentiation of passaged SCs after only two passages [11]. Apart from being a relatively harsh enzymatic passaging reagent, trypsin leads to the detachment of all the cells in the dish including SCs that are already committed to differentiate into myotubes, as well as any contaminating cells, which might contribute towards the loss of SC proliferative potential and accelerated differentiation. Indeed, even a gentler detachment solution like Accutase leads to loss of myogenic proliferative properties and accelerates differentiation. By contrast, the ICT approach favors the detachment of only the SCs that have not yet committed to differentiate, and in particular the slow dividing population which has previously been shown to retain stemness and long-term self-renewal ability [31]. It is conceivable that being 'true' stem cells, the slow dividing SCs detaching in response to cold temperature are the so-called first responders to stress or injury in vivo [5], a hypothesis that will be the subject of future investigation in our laboratory.

In a recent study, Gregory WC et al. demonstrated that hSCs differentiate and lose their proliferative potential when maintained in high mitogen conditions ex vivo. They used inhibition of p38 signalling to prevent the differentiation of SCs and promote their expansion [35]. Using our method, we were able to achieve the same but with minimal manipulation, maintaining the proliferative capacity of hSC ex vivo for an extended period of time, to a similar degree using muscle biopsies taken from a wide range of ages (between 50 and 90 years old). This is an important technical advance for both basic and clinical research since it will allow researchers to obtain sufficient number of cells for transplantation or intervention studies, while reducing the number of human biopsies required. Indeed, a major obstacle to stem cell-based therapies has been the scarcity of human muscle tissue specimens and the limited number of cells that can be obtained for transplantation.

Moreover, successful transplantation requires the use of freshly isolated SCs because culturing and expanding them in vitro greatly reduces their engraftment capacity [13]. Here, we show that SCs isolated and expanded using the ICT method do not lose their regenerative capacity.

Another advantage of the ICT method is the improved longevity of cultured myotubes. Generally, SCs differentiate into myotubes by day 7 of culture and shortly after, begin to detach [11,12]. Notably, SCs isolated and passaged with the ICT method could be maintained in culture for up to 2 months, even once they have differentiated into myotubes, likely due to the lack of contaminating cells such as fibroblasts. The purity of the isolated SCs is of paramount importance for in vitro studies since even 97% purity is insufficient to prevent overgrowth by non-myogenic cells, as we demonstrated. While important for cell growth, growth factors produced by fibroblasts, have been linked to senescence induction in long term cultures of mesenchymal stem cells [36].

(See figure on previous page.) Fig. 3 Isolation and in vitro expansion of human satellite cells using the ICT method. A. Representative bright field images of ICT-isolated human SCs following 1, 3, 5, and 10 ICTs, at day 3 and 10 of culture in GM, and at day 10 of culture in DM (n = 15 independent experiments). b Number of human SCs at 24, 48, and 72 h following ICT 1. c Total number of human SCs at day 3 of culture in GM after 1 and 10 ICTs. d Representative immunofluorescence images of human SCs stained for Pax7 (red) and nuclei (blue). Graph shows percentage of cells positive for Pax7 at day 2 of culture in GM. e Representative immunofluorescence images of human SCs stained for myogenin (red) and nuclei (blue). Graph shows percentage of cells positive for myogenin at day 5 of culture in GM. f Representative immunofluorescence images of human SCs stained for desmin (red) and nuclei (blue) after differentiation (10 days in GM + 5 days in DM). g Representative immunofluorescence images of human SCs stained for myosin heavy chain (MHC) (red) and nuclei(blue). Graph shows percent fusion after differentiation (10 days GM + 10 days DM). (n = 3 independent experiments, 10 images analyzed per experiment) Scale bar = 100 μm. Error bars represent mean ± sem

Conclusions

In the quest for new and improved SC isolation methods, the ideal technique would permit the isolation of pure SCs with minimal manipulation, that can be expanded ex vivo without losing their stemness and regenerative capacity. In terms of purity of the isolated cell population, the ICT method outperforms others such as the pre-plating method or the magnetic beads isolation method. Compared to other commonly used methods, it is fast and easy to perform, and apart from the time required for enzymatic digestion (1.5 h), it involves minimal manipulation of the cells. Finally, using the ICT approach, SCs can be expanded for extended periods of time without losing their proliferation and differentiation potential. This in turn drastically reduces the number of mice or muscle biopsies required to obtain sufficient number of cells.

Overall, the cost-effectiveness, accessibility and technical simplicity of this method, as well as its remarkable efficiency, represent major improvements over existing methods, and will no doubt accelerate SC basic and translational research bringing their therapeutic use closer to the clinic. Finally, this is a proof of concept study, and the ICT method can be further optimised, adapted, and improved for use in different experimental settings.

min RT, and incubated overnight at 4°C in 4% BSA in PBS with the following primary antibodies: mouse anti-Pax7-c (1:10 DSHB, Iowa City, IA, USA), rabbit anti-MyoD (1:50 Santa Cruz C20: sc-304, Dallas, TX, USA), mouse anti-myogenin (1:20, DSHB), mouse anti-Myosin Heavy Chain (1:20 DSHB), and mouse anti-desmin (1:20, DSHB). The next day, SCs were washed 3 times in PBS for 15 min, and then incubated with secondary antibodies goat anti-rabbit Alexa Fluor 488 (1:1000, Abcam) and goat anti-mouse Alexa Fluor 555 (1:1000, Thermo Fisher Scientific) diluted in 1% BSA in PBS, for 1 h RT. Nuclei were counterstained with Hoechst.

Fig. 1 (
1See legend on next page.)

Fig. 2 (
2See legend on next page.)

Fig. 3 (
3See legend on next page.)
AcknowledgementsWe thank Dr. Luca Madaro for critical reading of the manuscript and helpful discussions.Authors' contributions BLO conceived, developed, and standardized the method. BLO, AB, and MB designed the experiments. AB performed experiments. AB and BLO analyzed data and prepared the figures. BLO and MB supervised the study. CG, DMD, and VC provided the human muscle biopsy specimens. BLO wrote the manuscript. All authors critically read, edited, and approved the final manuscript.FundingThis work was supported by a grant from The Dutch Duchenne Parent Project NL (DPP NL) to BLO; research grants from Parent Project Italy (PP, Italy) to MB and from the University of Rome (# RP11715C7D238352, RM118164275C7EBE, and RM11916B7E20311C to MB; and # AR11715C7F9E158E, AR11816436905518, and AR11916B7E2A7B64 to AB).Availability of data and materialsAll data generated or analyzed during this study are included in this published article [and its supplementary information files]. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.DeclarationsEthics approval and consent to participate All procedures involving mice were approved by the Italian Ministry for Health and were conducted according to the EU regulations and the Italian Law on Animal Research. According to the Italian law, the authors are not required to ask for approval from an institutional review board or ethical committee for the use of human tissue removed during surgical procedures. All patients gave their approval to undergo intraoperative muscle biopsy and to publish the clinical and laboratory data obtained.Supplementary InformationThe online version contains supplementary material available at https://doi. org/10.1186/s13395-021-00261-w.Additional file 1:Figure S1. Myogenic properties of SCs isolated with the ICT method. A. Percent of myogenic colony formation was calculated as percent growing clones out of the total seeded single cells per well (60 per 96-well plate) among ICT-and MB-isolated SCs (n=3 independent experiments). B. Number of cells per clone in single clone-derived ICTand MB-isolated SCs at 48 and 72 h of culture in GM. ICT SCs, n= 37 clones analyzed per experiment. MB SCs, n= 37 clones analyzed per experiment. C. Percent of ICT-and MB-isolated SCs positive for Pax7 at day 2 of culture in GM. (n=3 independent experiments). D. Percent fusion of single clone-derived ICT and MB-isolated SCs after differentiation (4 days in GM followed by 3 days in DM). Fusion index: number of nuclei within myotubes divided by total number nuclei. E. Number of nuclei per myotube in single clone-derived ICT and MB-isolated SCs after differentiation (4 days in GM followed by 3 days in DM). (n=3 independent experiments). Error bars represent mean ± sem.Additional file 2:Figure S2. Increased longevity in culture of ICTisolated SCs. A. Representative bright field images of ICT-isolated SCs at day 3, 5, 11 and 17 of culture in DM. B. Representative bright field image of MB isolated SCs at day 3 of culture in DM. C. Total number of days in culture of ICT-and MB-isolated SCs. D. Representative bright field images showing the heterogeneous muscle cell culture after pre-plating, at 3 and 5 days of culture in GM. Error bars represent mean ± sem. *P < 0.05 by Student's t-test.Additional file 3:Figure S3. In vivo functional validation of SCs isolated using the ICT and MB method. A. Representative immunofluorescence images of dystrophin positive fibers (green) and nuclei (blue) in mdx tibialis muscle at 30 days following intra-muscular injection of 15 x 10 3 SCs immediately after ICT isolation or after 3 dayexpansion in culture following ICT isolation. B. Quantification of the number of dystrophin positive fibers per TA muscle section in mdx mice (ICT, n=5 mdx mice; ICT-expanded, n=4 mdx mice). Scale bar=100μm. Error bars represent mean ± sem. C. Representative immunofluorescence images of dystrophin positive fibers (green) and nuclei (blue) in mdx tibialis muscle at 30 days following intra-muscular injection of 15 x 10 3 ICTisolated SCs (left), or MB-freshly isolated SCs (right). B. Quantification of the number of dystrophin positive fibers per TA section in mdx mice (ICT, n=5 mdx mice; MB, n=5 mdx mice). Scale bar=100μm. Error bars represent mean ± sem.Additional file 4:Figure S4. Characterisation of the human musclederived cells obtained after pre-plating and prior to ICT. A. Representative bright field images of human muscle-derived cells at day 3 and 10 of culture in GM, and at day 10 of culture in DM. B. Representative immunofluorescence images of the heterogeneous culture of human musclederived cells stained for Pax7 (red) and nuclei (blue). Graph shows percentage of cells positive for Pax7 at day 2 of culture in GM. C. Representative immunofluorescence images of the heterogeneous human muscle cell culture stained for myogenin (red) and nuclei (blue). Graph shows percent of cells positive for myogenin at day 5 of culture in GM. D. Representative immunofluorescence images of the heterogeneous culture of human muscle-derived cells stained for MHC (red) and nuclei (blue). Graph shows percent cell fusion after differentiation (10 days in GM followed by 10 days in DM). E. Graph shows percent myogenic cells in the human heterogenous muscle cells obtained after pre-plating or after ICT at day 5 of culture in GM, calculated by IF staining for myogenin (n= 3 independent experiments, 10 images analysed per experiment). Scale bar=100μm. Error bars represent mean ± sem. ****P < 0.0001 by Student's t-test.Consent for publicationNot applicable.Competing interestsThe authors declare that they have no competing interests.Author details1
Satellite Cells: The Architects of Skeletal Muscle. N C Chang, M A Rudnicki, Curr Top Dev Biol. 107Chang NC, Rudnicki MA. Satellite Cells: The Architects of Skeletal Muscle. Curr Top Dev Biol. 2014;107:161-81.

Muscle stem cells at a glance. Y X Wang, N A Dumont, M A Rudnicki, J Cell Sci. 127Wang YX, Dumont NA, Rudnicki MA. Muscle stem cells at a glance. J Cell Sci. 2014;127:4543-8.

Satellite cell of skeletal muscle fibers. A Mauro, J Biophys Biochem Cytol. 9Mauro A. Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol. 1961;9:493-5.

Satellite cells, the engines of muscle repair. Y X Wang, M A Rudnicki, Nat Rev Mol Cell Biol. 13Wang YX, Rudnicki MA. Satellite cells, the engines of muscle repair. Nat Rev Mol Cell Biol. 2012;13:127-33.

Skeletal muscle stem cells in comfort and stress. B Evano, S Tajbakhsh, Regen Med. 324Evano B, Tajbakhsh S. Skeletal muscle stem cells in comfort and stress. npj Regen Med. 2018;3:24.

Muscle stem cells. P Feige, M A Rudnicki, Curr Biol. 28Feige P, Rudnicki MA. Muscle stem cells. Curr Biol. 2018;28:581-98.

Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration. R Sambasivan, R Yao, A Kissenpfennig, L Van Wittenberghe, A Paldi, B Gayraud-Morel, Development. 138Sambasivan R, Yao R, Kissenpfennig A, van Wittenberghe L, Paldi A, Gayraud-Morel B, et al. Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration. Development. 2011;138:3647-56.

Satellite cells are essential for skeletal muscle regeneration: The cell on the edge returns centre stage. Development (Cambridge). F Relaix, P S Zammit, 139Relaix F, Zammit PS. Satellite cells are essential for skeletal muscle regeneration: The cell on the edge returns centre stage. Development (Cambridge). 2012;139:2845-56.

Repairing skeletal muscle: Regenerative potential of skeletal muscle stem cells. F S Tedesco, A Dellavalle, J Diaz-Manera, G Messina, G Cossu, J Clin Investig. 120Tedesco FS, Dellavalle A, Diaz-Manera J, Messina G, Cossu G. Repairing skeletal muscle: Regenerative potential of skeletal muscle stem cells. J Clin Investig. 2010;120:11-9.

Isolation and culture of skeletal muscle myofibers as a means to analyze satellite cells. P Keire, A Shearer, G Shefer, Z Yablonka-Reuveni, Methods Mol Biol. 946Keire P, Shearer A, Shefer G, Yablonka-Reuveni Z. Isolation and culture of skeletal muscle myofibers as a means to analyze satellite cells. Methods Mol Biol. 2013;946:431-68.

Skeletal muscle satellite cells: Background and methods for isolation and analysis in a primary culture system. M E Danoviz, Z Yablonka-Reuveni, Methods Mol Biol. 798Danoviz ME, Yablonka-Reuveni Z. Skeletal muscle satellite cells: Background and methods for isolation and analysis in a primary culture system. Methods Mol Biol. 2012;798:21-52.

Isolation and Purification of Satellite Cells for Skeletal Muscle Tissue Engineering. D Jonah, Bcs Lee, M Lisa, Kwv Larkin, J Regen Med. 3117Jonah D, Lee BCS, Lisa M, Larkin KWV. Isolation and Purification of Satellite Cells for Skeletal Muscle Tissue Engineering. J Regen Med. 2015;3:117.

Stem cells for skeletal muscle regeneration: Therapeutic potential and roadblocks. F Rinaldi, Rcr Perlingeiro, Transl Res. Rinaldi F, Perlingeiro RCR. Stem cells for skeletal muscle regeneration: Therapeutic potential and roadblocks. Transl Res. 2014.

Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique. B Gharaibeh, A Lu, J Tebbets, B Zheng, J Feduska, M Crisan, Nat Protoc. 163Gharaibeh B, Lu A, Tebbets J, Zheng B, Feduska J, Crisan M, et al. Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique. Nat Protoc. 2008;163:409-17.

A Modified Pre-plating Method for High-Yield and High-Purity Muscle Stem Cell Isolation From Human/Mouse Skeletal Muscle Tissues. K Yoshioka, Y Kitajima, N Okazaki, K Chiba, A Yonekura, Y Ono, Front Cell Dev Biol. 8793Yoshioka K, Kitajima Y, Okazaki N, Chiba K, Yonekura A, Ono Y. A Modified Pre-plating Method for High-Yield and High-Purity Muscle Stem Cell Isolation From Human/Mouse Skeletal Muscle Tissues. Front Cell Dev Biol. 2020;8:793.

Isolation of muscle stem cells by fluorescence activated cell sorting cytometry. A Pasut, P Oleynik, M A Rudnicki, Methods Mol Biol. 798Pasut A, Oleynik P, Rudnicki MA. Isolation of muscle stem cells by fluorescence activated cell sorting cytometry. Methods Mol Biol. 2012;798: 53-64.

Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting. L Liu, T H Cheung, G W Charville, T A Rando, Nat Protoc. 10Liu L, Cheung TH, Charville GW, Rando TA. Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting. Nat Protoc. 2015;10:1612-24.

Sorting single satellite cells from individual myofibers reveals heterogeneity in cell-surface markers and myogenic capacity. M R Chapman, K R Balakrishnan, J Li, M J Conboy, H Huang, S K Mohanty, Integr Biol (United Kingdom). 5Chapman MR, Balakrishnan KR, Li J, Conboy MJ, Huang H, Mohanty SK, et al. Sorting single satellite cells from individual myofibers reveals heterogeneity in cell-surface markers and myogenic capacity. Integr Biol (United Kingdom). 2013;5:692-702.

Purification and cell-surface marker characterization of quiescent satellite cells from murine skeletal muscle by a novel monoclonal antibody. S I Fukada, S Higuchi, M Segawa, K I Koda, Y Yamamoto, K Tsujikawa, Exp Cell Res. 296Fukada SI, Higuchi S, Segawa M, Koda KI, Yamamoto Y, Tsujikawa K, et al. Purification and cell-surface marker characterization of quiescent satellite cells from murine skeletal muscle by a novel monoclonal antibody. Exp Cell Res. 2004;296:245-55.

Isolation of adult mouse myogenic progenitors: Functional heterogeneity of cells within and engrafting skeletal muscle. R I Sherwood, J L Christensen, I M Conboy, M J Conboy, T A Rando, I L Weissman, Cell. 119Sherwood RI, Christensen JL, Conboy IM, Conboy MJ, Rando TA, Weissman IL, et al. Isolation of adult mouse myogenic progenitors: Functional heterogeneity of cells within and engrafting skeletal muscle. Cell. 2004;119: 543-54.

Direct Isolation of Satellite Cells for Skeletal Muscle Regeneration. D Montarras, J Morgan, C Collins, Mol Cell Biol. 309Montarras D, Morgan J, Collins C. Direct Isolation of Satellite Cells for Skeletal Muscle Regeneration. Mol Cell Biol. 2005;309:2064-7.

Purification of mouse primary myoblasts based on α7 integrin expression. W E Blanco-Bose, C C Yao, R H Kramer, H M Blau, Exp Cell Res. 265Blanco-Bose WE, Yao CC, Kramer RH, Blau HM. Purification of mouse primary myoblasts based on α7 integrin expression. Exp Cell Res. 2001;265:212-20.

Targeting pkcθ promotes satellite cell self-renewal. A Benedetti, P F Fiore, L Madaro, B Lozanoska-Ochser, M Bouché, Int J Mol Sci. 212419Benedetti A, Fiore PF, Madaro L, Lozanoska-Ochser B, Bouché M. Targeting pkcθ promotes satellite cell self-renewal. Int J Mol Sci. 2020;21:2419.

Lack of PKCθ promotes regenerative ability of muscle stem cells in chronic muscle injury. P F Fiore, A Benedetti, M Sandonà, L Madaro, M De Bardi, V Saccone, Int J Mol Sci. 21932Fiore PF, Benedetti A, Sandonà M, Madaro L, de Bardi M, Saccone V, et al. Lack of PKCθ promotes regenerative ability of muscle stem cells in chronic muscle injury. Int J Mol Sci. 2020;21:932.

Splenic Ly6Chi monocytes are critical players in dystrophic muscle injury and repair. G Rizzo, R Di Maggio, A Benedetti, J Morroni, M Bouche, B Lozanoska-Ochser, JCI Insight. 5130807Rizzo G, di Maggio R, Benedetti A, Morroni J, Bouche M, Lozanoska-Ochser B. Splenic Ly6Chi monocytes are critical players in dystrophic muscle injury and repair. JCI Insight. 2020;5:e130807.

Targeting early PKCθ-dependent T-cell infiltration of dystrophic muscle reduces disease severity in a mouse model of muscular dystrophy. B Lozanoska-Ochser, A Benedetti, G Rizzo, V Marrocco, R Di Maggio, P Fiore, J Pathol. 244Lozanoska-Ochser B, Benedetti A, Rizzo G, Marrocco V, di Maggio R, Fiore P, et al. Targeting early PKCθ-dependent T-cell infiltration of dystrophic muscle reduces disease severity in a mouse model of muscular dystrophy. J Pathol. 2018;244:323-333.

The adhesion of Chinese hamster cells. I. Effects of temperature, metabolic inhibitors and proteolytic dissection of cell surface macromolecules. R L Juliano, E Gagalang, J Cell Physiol. 92Juliano RL, Gagalang E. The adhesion of Chinese hamster cells. I. Effects of temperature, metabolic inhibitors and proteolytic dissection of cell surface macromolecules. J Cell Physiol. 1977;92:209-20.

Temperature modulation of integrin-mediated cell adhesion. F Rico, C Chu, M H Abdulreda, Y Qin, V T Moy, Biophys J. 99Rico F, Chu C, Abdulreda MH, Qin Y, Moy VT. Temperature modulation of integrin-mediated cell adhesion. Biophys J. 2010;99:1387-96.

Primary rat muscle progenitor cells have decreased proliferation and myotube formation during passages. S Machida, E E Spangenburg, F W Booth, Cell Prolif. 37Machida S, Spangenburg EE, Booth FW. Primary rat muscle progenitor cells have decreased proliferation and myotube formation during passages. Cell Prolif. 2004;37:267-77.

Satellite Cell Heterogeneity in Skeletal Muscle Homeostasis. M T Tierney, A Sacco, Trends Cell Biol. 26Tierney MT, Sacco A. Satellite Cell Heterogeneity in Skeletal Muscle Homeostasis. Trends Cell Biol. 2016;26:434-444.

Slowdividing satellite cells retain long-term self-renewal ability in adult muscle. Y Ono, S Masuda, H S Nam, R Benezra, Y Miyagoe-Suzuki, S Takeda, J Cell Sci. 125Ono Y, Masuda S, Nam HS, Benezra R, Miyagoe-Suzuki Y, Takeda S. Slow- dividing satellite cells retain long-term self-renewal ability in adult muscle. J Cell Sci. 2012;125:1309-17.

Ex vivo expansion and in vivo self-renewal of human muscle stem cells. G W Charville, T H Cheung, B Yoo, P J Santos, G K Lee, J B Shrager, Stem Cell Reports. 5Charville GW, Cheung TH, Yoo B, Santos PJ, Lee GK, Shrager JB, et al. Ex vivo expansion and in vivo self-renewal of human muscle stem cells. Stem Cell Reports. 2015;5:621-32.

Human satellite cells have regenerative capacity and are genetically manipulable. A Marg, H Escobar, S Gloy, M Kufeld, J Zacher, A Spuler, J Clin Investig. 124Marg A, Escobar H, Gloy S, Kufeld M, Zacher J, Spuler A, et al. Human satellite cells have regenerative capacity and are genetically manipulable. J Clin Investig. 2014;124:4257-65.

Molecular Biology of Thermoregulation Invited Review: Effects of heat and cold stress on mammalian gene expression. L Sonna, J Fujita, S L Gaffin, M Craig, J Appl Physiol. 92Sonna L, Fujita J, Gaffin SL, Craig M. Molecular Biology of Thermoregulation Invited Review: Effects of heat and cold stress on mammalian gene expression. J Appl Physiol. 2002;92:1725-42.

Inhibition of Methyltransferase Setd7 Allows the In Vitro Expansion of Myogenic Stem Cells with Improved Therapeutic Potential. R N Judson, M Quarta, M J Oudhoff, H Soliman, L Yi, C K Chang, Cell Stem Cell. 22Judson RN, Quarta M, Oudhoff MJ, Soliman H, Yi L, Chang CK, et al. Inhibition of Methyltransferase Setd7 Allows the In Vitro Expansion of Myogenic Stem Cells with Improved Therapeutic Potential. Cell Stem Cell. 2018;22:177-90.

FGF-2 suppresses cellular senescence of human mesenchymal stem cells by down-regulation of TGF-β2. T Ito, R Sawada, Y Fujiwara, Y Seyama, T Tsuchiya, Biochem Biophys Res Commun. 359Ito T, Sawada R, Fujiwara Y, Seyama Y, Tsuchiya T. FGF-2 suppresses cellular senescence of human mesenchymal stem cells by down-regulation of TGF- β2. Biochem Biophys Res Commun. 2007;359:108-14.

Publisher's Note. Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Post-Critique, Politics, and the Political in Educational Philosophy

Naomi Hodgson
Liverpool Hope University

Post-Critique, Politics, and the Political in Educational Philosophy
10.17899/on_ed.2020.9.3_vol. 3_issue # 9 1affirmativeessencepoliticalpost-critiquesurface
The recent Manifesto for a Post-Critical Pedagogy introduced a specifically pedagogical register into theoretical and methodological consideration of post-critique. Focusing on a specific aspect of the Manifesto -the view that the political concerns of much critical educational research position education as instrumental to politics to the extent that the "educational" in educational research is left out of the picture -I ask to what extent we can defend the view that education and politics should be separate in our enquiries? Drawing on a particular account of the separation of education and politics I suggest that what is at issue is not the political as such but the particular, sociological, register of politics at work in critical educational research. To bracket out the political is potentially to leave everyday flesh and blood experiences of education out of the picture.

The notion of the post-critical has gained traction in a number of fields in recent years, from literary studies to social sciences to philosophy, in work that has questioned the epistemological and ontological force of critique. In literary studies, for example, Frank and Sedgwick (1995), Anker and Felski (2017) and others have sought to move away from the "hermeneutics of suspicion" that has characterised critical textual analysis. In the field of social sciences, Latour (2004Latour ( , 2005 has questioned the ability of critique to gather publics, and what its current form means for the relationship between science and democracy. While in both cases we see a shift away from a particular form of political reading, politics remains part of the picture, of what we are addressing, why, and how, as academics.

The post-critical has been given more recent articulation in the field of education in the Manifesto for a Post-Critical Pedagogy (Hodgson et al., 2017), which sought to make manifest a more general sense in the field of educational philosophy of a need to move beyond the political emphasis of much critical educational research. Influenced by Latour, Arendt, and more literary influences on the field such as Cavell and Emerson, the Manifesto sets out five principles for a post-critical attitude to educational inquiry that seeks "not to debunk but to protect and to care". Echoing work in literary studies, we proposed a shift from the hermeneutical pedagogy that we associate with critical pedagogy, to pedagogical hermeneutics, based on "the assumption that we can speak and act -together" (Hodgson et al., 2017, p. 18):

It is precisely the challenges of living together in a common world that constitute the hope that make education continue to seem a worthwhile activity. Hermeneutics isn't a (unsolvable) problem, but rather something educators need to create. We shouldn't speak and act on the basis of a priori assumptions about the (im)possibility of real mutual understanding and respect, but rather show that, in spite of the many differences that divide us, there is a space of commonality that only comes about a posteriori (cf. Arendt, Badiou, Cavell). (Hodgson et al., 2017, p. 18) By invoking hope in the present, we sought to articulate an affirmative mode of educational inquiry, focused not (or not only) on critique of the dysfunction of our institutions but on reclaiming "the suppressed parts of our experience" (Hodgson et al., 2017, p. 19; I will return to what is implied by "suppressed" here later). The invocation of an Arendtian love for the world meant not an acceptance of how things are, but an affirmation of the value of what we do in the present and thus of things that we value as worth passing on. But not as they are: educational hope is about the possibility of a renewal of our common world. (Hodgson et al., 2017, p. 18) In this essay I focus on a specific aspect of the Manifesto -the view that the political concerns of much critical educational research position education as instrumental to politics to the extent that the "educational" in educational research is left out of the picture -to ask: Even if we accept that critical accounts, which seek to release education from its fulfilment of economic policy or reproduction of inequality, only reconceive it in terms of other political aims, to what extent can we defend the view that education and politics should be separate in our enquiries?

This has been a year distinguished in its awfulness: environmental disasters including wildfires and floods, the killing of black citizens by police in the US, and a pandemic that has brought existing inequalities and prejudices into sharp relief. School strikes, the Black Lives Matter movement, providing education to children and young people both at home and at school, reopening amid ongoing social distancing and enhanced health and safety measures, and caring for those struggling, grieving, and anxious when returning to formal education: educational institutions and professionals have had no choice but to respond and adapt to each cause and crisis. Discussion of education today, then, whether public or academic, seems unavoidably political. So, what form do recent interventions that argue, following Arendt, that the defence of education requires strict separation of politics and education (where education usually refers to schooling) take and what affirmative force do they have?

The notion of the school as separate from politics is a radical move: from a critical theoretical perspective, the school is political all the way down, from the content of the curriculum, the diversity of the teaching staff, the pedagogies, policies, and aims; from the perspective of those designing policy, there are wider socioeconomic aims to which schools must contribute; for those implementing policy on a local level, their success is measured on their ability to foster an inclusive learning environment and to 'close the gap' between those from disadvantaged backgrounds and the highest achieving, and so on. But this separation is a position that guides those in power themselves: the notion of equal opportunities relies on the idea that where you are born should not determine educational outcomes and therefore all children should receive the same standard of access to education. The differences in outcomes can then be attributed to individual school failings, or to individuals' lack of aspiration, grit, and resilience. We should be careful, then -particularly at this time when certain aspects of public discourse are prone to write off any appeal to identity categories as 'woke', 'political correctness' or 'left-wing bias' -how we define and articulate the political as it relates to education. Given the year that 2020 has been and the longer term challenges we face, it seems counter-intuitive perhaps to insist on the separation of education and politics. How education is governed, who it excludes, how it prepares our children for the future, and so on are pressing concerns, as any newspaper or indeed academic call for papers published since March 2020 will attest.

The argument for education and politics as appropriately separate spheres is strongly articulated by Joris Vlieghe and Piotr Zamojski. The ontological turn in their work effects the affirmative attitude called for by post-critical pedagogy by focusing in on the essence of education. In their 2019 book Towards an Ontology of Teaching: Thing-centred Pedagogy, Affirmation and Love for the World, Vlieghe and Zamojski investigate what "teaching consists [of] essentially" (p. 58) and "what education essentially is" (p. 73). In a 2020 article, they set out their position in relation to the 'critical' position that is concerned with recognising the inevitable political dimension of education. As they characterise it: either it needs to be revealed to those who don't realise its political function or, once it has been revealed, education needs to work in service of emancipation from this status quo. For Vlieghe and Zamojski, the distinction between education and politics is not a denial that there is anything political about education, rather it is a reorienting of critique from being predominantly concerned with politics at the expense of the educational. Relentless radical critique is, they argue, self-defeating (Vlieghe & Zamojski, 2020, p. 3). As Rancière (1991) has argued, "the very idea that we, as educators, first need to enlighten the oppressed other about her oppressive conditions, installs a regime of inequality we can never escape from" (Vlieghe & Zamojski, 2020, p. 3).

Vlieghe and Zamojski draw on Arendt's (1958Arendt's ( , 1961 distinction between education and politics as two separate spheres, and they further distinguish between the two senses of politics she offers: the strong sense and the wrong sense. The former refers to: the activity of those who leave the private sphere behind, in order to gather on the agora, to speak and discuss, and so to perform deeds through which a new beginning can be introduced to our common world. Politics in the strong sense is therefore a practice of establishing the common (Vlieghe & Zamojski, 2020, p. 4).

Politics "in the wrong sense", on the other hand, is illustrated in the contemporary politicisation of education, in which education acts a means to realise "a political dream that exploits and sacrifices the newness of the new generation to fulfil the fantasy of the perfect society" (Vlieghe & Zamojski, 2020, p. 4). The argument for the need to separate education from politics is premised on the Arendtian understanding of education as "a response to newness", which gives "the newcomers the possibility to begin anew with this world" (Vlieghe & Zamojski, 2020, p. 4). What makes this passing on of the old world to the new generation "properly humane and educational is that this gift should be passed on in such a way that the new generation can actually take up this world in ways we, the adults, did not and could not have foreseen" (Vlieghe & Zamojski, 2020, p. 4).

Vlieghe and Zamojski restate the idea (see Hodgson et al., 2017Hodgson et al., , 2018) that critical work is premised on hate: On the basis that the world is assumed [by the critic] to be evil, the stance of this master of critique is driven by a certain hate for the existing form of the world, and simultaneously by love for an imaginary world that will never come. (Vlieghe & Zamojski, 2020, p. 10) This supports the link between an affirmative approach and an inherently educational one:

We take our cue here, again, from Arendt, who concludes her essay on the crisis in education with the assertion that education essentially consists of displaying love for the world, which suggests (although Arendt proposed an alternative argument when developing the strong sense of politics in her political writings, e.g. Arendt, 1958) that politics is predicated on hate for the world (Vlieghe & Zamojski, 2020, p. 5).

Vlieghe and Zamojski nuance the idea that politics is driven by hate and education by love in two ways: first, in relation to an ontological conception of love and hate, and second in relation to Agamben's notion of potentiality.

In relation to the first, to conceive of love and hate ontologically they draw on Scheler's (1973) conception of love and hate as "two fundamental modes of world disclosure" (Vlieghe & Zamojski, 2020, p. 5). Love "is a way of relating to the world that allows the world to open up (and hence to be affirmed as worthwhile), which invites us to explore and study it, and to pass it on to the next generations" (Vlieghe & Zamojski, 2020, p. 5). Hate narrows down the world (which is, phenomenologically speaking, a precise description of the manner in which the world appears to a political activist: there is something in the world that is so wrong that one simply needs to correct this wrong rather than putting one's energy into anything else) (Vlieghe & Zamojski, 2020, p. 5).

The references to "hate" (Hodgson et al., 2017(Hodgson et al., , 2018 and to "evil" in later work by Vlieghe and Zamojski (2020) refer to the reliance of the critical pedagogue on the very unequal status quo from which they seek to emancipate the other and their standing in judgment of it from outside. To be driven by a passion of hate -of injustice, of inequality -seems not inherently bad, however; the point is that "relentless radical critique" (Vlieghe & Zamojski, 2020, p. 3) risks becoming an almost myopic focus whereby the possibility of education is denied, such is the belief in the evil of the system. Articulated in Arendtian terms:

To formulate more positively the role of the pedagogue as initiating the new generation into a common world, we offer the idea of a post-critical pedagogy, which requires a love for the world. This is not an acceptance of how things are, but an affirmation of the value of what we do in the present and thus of things that we value as worth passing on (Hodgson et al., 2017, p. 18).

In relation to the second way in which they nuance the account, they draw on Agamben's notion of potentiality to situate Arendt's insistence on the autonomy of education: because study comes with an experience and sense of potentiality, it also makes possible political thought and action. At the risk of making a truly paradoxical statement, one must first experience pure potentiality in the autonomous, separate sphere of education, to be able to intervene in the current order of things in politics. In that sense, education is not just an end in itself, but precisely a pure means: it could be instrumental for politics (which explains why it can be so easily instrumentalised), but it can only be of use on the condition that it remains fully in potentiality and that this political potential does not get actualised (Vlieghe & Zamojski, 2020, p. 11).

The bracketing out of politics in order that the pedagogical realm is unencumbered by the fantasies of the older generation, however, seems compromised by the tone in which Vlieghe and Zamojski present the binaries of love and hate, the teacher and the activist. Love, in the Arendtian formulation above, is articulated in terms of what we do in our pedagogical relationships that constitute that initiation, and our judgments on and investments in what we value as worth passing on. In expressing a concern with what we value this seems to inherently and unavoidably entail the political. This separation of politics and education in view of essences also seems to imply the bracketing out of subjectivity, as if a subject matter could be taught and learned without our very subjectivity sitting between us and the world.

The argument that politics is seen to detract from, supercede, or determine educational concerns in critical pedagogy and critical educational research more generally as it is made by Vlieghe and Zamojski overlooks that a particular register of politics is at work: a predominantly sociological one that informs much educational research that is ordered around identity categories, such as race and gender, and institutions, e.g. school, university, government. Taking this into account, the relationship between education and politics in the field of educational research makes sense. It is perhaps not 'politics' as such that is the problem but the conception of it that potentially closes down educational aspects. This sociological register of politics as it appears in the critical educational literature is left unquestioned in Vlieghe and Zamojski's article in that they focus not on the conception of the political at work in critical pedagogy but the way it impinges on education.

As suggested earlier, part of the affirmative aspect of the post-critical entails the will to articulate that which is left out of the picture, not only by dominant theoretical and methodological approaches but also prevailing discourses. Placing strict conditions on what education and politics are and can be, however, seems to narrow down the picture of both. It is possible to resist the instrumentalist overdetermination of school education (and the account Vlieghe and Zamojski offer does seem limited to school education) without denying the inherent existence of the political in the educational (and vice versa). What does it mean for our work to consider the relationship between the educational and the political more broadly rather than restricting our articulation of new approaches to a binary of school education and a sociological conception of politics? (For one possible answer see Standish, 2019).

The force of Arendt's argument for the separation of education and politics (briefly, that the political concerns of modern education "insofar as it attempts to establish a world of children, destroys the necessary conditions for vital development and growth" (Arendt, 1953, p. 8)) lies in part in its call to test ourselves: to what extent are we taking responsibility as the older generation for present problems?

To what extent are we predetermining who we want our children to be? To what extent are my fears for the future rooted in my sense of lack of control of the present? These are not only academic questions. They reflect the very concerns -mundane and existential -we face each day as parents, educators, grown-ups.

A strong impetus of critical educational research is the resistance of forms of governance and subjectivation that entrench or constitute inequity due to discrimination and injustice. The turn to the post-critical seeks to reanimate the possibility of critique in this sense, not through an attitude of suspicion and debunking but of affirming that which we value and of articulating that which is left out of the picture. Concerned that Vlieghe and Zamojski's focus on essences is potentially a totalitarian move, Kai Wortmann goes in "the opposite direction: not reaching down to the deep essences but rather staying on the surface of things" (2019, p. 475). I take Wortmann's approach as a call not to restrict ourselves to the merely superficial, but rather as a concern for the mundane, and with what we can see. This does not imply an analysis that lacks depth, nor one that seeks essences, at least in the sense that Vlieghe and Zamojski invoke. 1 In the context of discussions of the post-critical, the invocation of surface and depth has a particular resonance. It is precisely such topological accounts of reading, those that offer a suspicious reading that reveals "hidden ideology … the workings of power" (Moi, 2017, p. 175), that post-critique in literary theory sought to move away from. A similar move is proposed by post-critical pedagogy -towards an attitude that does not seek to debunk but to affirm. But Vlieghe and Zamojski offer a reading that is explicit in its suspicion of politics and that seeks to reveal the essence of education and teaching that has heretofore been concealed by it. Wortmann's appeal to attend to what is on the surface of things invokes a need to attend to our everyday practices and experience, bringing together the affirmative and the critical. In reference to the post-critical work of Latour, Anker and Felski, and Sedgwick, Wortmann notes that the new vocabularies they introduce "find hope not deep down but at the surface of things (Best & Marcus 2009)" (Wortmann, 2019, p. 476). The idea mentioned earlier of reclaiming "the suppressed parts of our experience" (Hodgson et al., 2017, p. 19) perhaps risks echoing the suspicion that informs critical readings. In an affirmative vein, however, I take it to refer to those aspects of our experience we cannot (yet) articulate, that are left out of the picture by dominant discourses and how they articulate our practices and their purposes, and the need to (re)establish "our relation to our words, opening them to question, and giving philosophical attention to these devalued aspects of our forms of life" (Hodgson et al., 2017, p. 17). By narrowing down education to essences and clipping the political -not politics in the sociological sense, but the political -out of the picture, the affirmative account of education is denied any sense of the tangible. It brackets out that which constitutes the flesh and blood of education.

Potentialities. Collected essays in philosophy. G Agamben, D. Heller-RoazenStanford University PressTrans.Agamben, G. (2000). Potentialities. Collected essays in philosophy. (D. Heller-Roazen, Trans.). Stanford University Press.

Introduction. E S Anker, R Felski, Critique and postcritique. E. S. Anker, & R. FelskiDuke University PressAnker, E. S., & Felski, R. (2017). Introduction. In E. S. Anker, & R. Felski (Eds.), Critique and postcritique (pp. 1-28). Duke University Press.

The human condition. H Arendt, University of Chicago PressArendt, H. (1958). The human condition. University of Chicago Press.

The crisis in education. H Arendt, H. Arendt, Between past and future: Eight exercises in political thought. Viking PressArendt, H. (1961). The crisis in education. In H. Arendt, Between past and future: Eight exercises in political thought (pp. 173-196). Viking Press.

Surface reading: An introduction. S Best, S Marcus, 10.1525/rep.2009.108.1.1Representations. 1081Best, S., & Marcus, S. (2009). Surface reading: An introduction. Representations, 108(1), 1-21. https://doi.org/10.1525/rep.2009.108.1.1

Shame in the cybernetic fold: Reading Silvan Tomkins. A Frank, E Sedgwick, Critical Inquiry. 212Frank, A., & Sedgwick, E. (1995). Shame in the cybernetic fold: Reading Silvan Tomkins. Critical Inquiry, 21(2), 496-522.

Manifesto for a post-critical pedagogy. punctum books. N Hodgson, J Vlieghe, P Zamojski, Hodgson, N., Vlieghe, J., & Zamojski, P. (2017). Manifesto for a post-critical pedagogy. punctum books. on_education Journal for Research and Debate _ISSN 2571-7855

. 10.17899/on_ed.2020.9.3_vol.3_issue#9_DOI 10.17899/on_ed.2020.9.3 _vol. 3_issue # 9

Education and the love for the world: Articulating a post-critical educational philosophy. N Hodgson, J Vlieghe, P Zamojski, 10.14516/fde.57616Foro de EducaciónHodgson, N., Vlieghe, J., & Zamojski, P. (2018). Education and the love for the world: Articulating a post-critical educational philosophy. Foro de Educación, 16(24), 7-20. https://doi.org/10.14516/fde.576

Philosophical presentations of raising children: The grammar of upbringing. N Hodgson, S Ramaekers, 10.1007/978-3-030-12540-0Palgrave MacmillanHodgson, N., & Ramaekers, S. (2019). Philosophical presentations of raising children: The grammar of upbringing. Palgrave Macmillan. https://doi.org/10.1007/978-3-030-12540-0

Why has critique run out of steam? From matters of fact to matters of concern. B Latour, Critical Inquiry. 302Latour, B. (2004). Why has critique run out of steam? From matters of fact to matters of concern. Critical Inquiry, 30(2), 225-248.

From Realpolitik to Dingpolitik or how to make things public. B Latour, Making things public: Atmospheres of democracy. B. Latour, & P. WeibelCenter for Art and MediaLatour, B. (2005). From Realpolitik to Dingpolitik or how to make things public. In B. Latour, & P. Weibel (Eds.), Making things public: Atmospheres of democracy (pp. 14-43). Center for Art and Media.

Revolution of the ordinary. T Moi, University of Chicago PressMoi, T. (2017). Revolution of the ordinary. University of Chicago Press.

The ignorant schoolmaster. J Rancière, Stanford University PressRoss, Trans.Rancière, J. (1991). The ignorant schoolmaster. (K. Ross, Trans.). Stanford University Press. (Original work published 1987)

Formalism in ethics and non-formal ethics of values. A new attempt toward the foundation of an ethical personalism. M Scheler, Trans.). M. S. Frings and R. L. Funk1913Northwestern University PressScheler, M. (1973). Formalism in ethics and non-formal ethics of values. A new attempt toward the foundation of an ethical personalism. (M. S. Frings and R. L. Funk, Trans.). Northwestern University Press. (Original work published 1913, 1916)

Paranoid reading and reparative reading, or, you're so paranoid, you probably think this essay is about you. E K Sedgwick, E. K. Sedgwick, Touching feeling: Affect, pedagogy, performativity. Duke University PressSedgwick, E. K. (2003). Paranoid reading and reparative reading, or, you're so paranoid, you probably think this essay is about you. In E. K. Sedgwick, Touching feeling: Affect, pedagogy, performativity (pp. 123-151). Duke University Press.

The educational is political. P Standish, 10.7571/esjkyoiku.13.5Educational Studies in Japan: International Yearbook. 13Standish, P. (2019). The educational is political. Educational Studies in Japan: International Yearbook, 13, 5-22. https://doi.org/10.7571/esjkyoiku.13.5

Towards an ontology of teaching. Thing-centred pedagogy, affirmation and love for the world. J Vlieghe, P Zamojski, 10.1007/978-3-030-16003-6SpringerVlieghe, J., & Zamojski. P. (2019). Towards an ontology of teaching. Thing-centred pedagogy, affirmation and love for the world. Springer. https://doi.org/10.1007/978-3-030-16003-6

Redefining education and politics: On the paradoxical relation between two separate spheres. J Vlieghe, P Zamojski, 10.1177/1478210320943808Policy Futures in Education. 187Vlieghe, J., & Zamojski, P. (2020). Redefining education and politics: On the paradoxical relation between two separate spheres. Policy Futures in Education, 18(7), 864-877. https://doi.org/10.1177/1478210320943808

Philosophical investigations. L Wittgenstein, Trans.). Basil Blackwell. G. E. M. AnscombeWittgenstein, L. (1953). Philosophical investigations. (G. E. M. Anscombe, Trans.). Basil Blackwell. (Original work published 1953)

Post-critical pedagogy as poetic practice: Combining affirmative and critical vocabularies. K Wortmann, 10.1080/17449642.2019.1669942Ethics and Education. 144Wortmann, K. (2019). Post-critical pedagogy as poetic practice: Combining affirmative and critical vocabularies. Ethics and Education, 14(4), 467-481. https://doi.org/10.1080/17449642.2019.1669942

Recommended Citation. Recommended Citation

Post-critique, politics, and the political in educational philosophy. N Hodgson, 10.17899/on_ed.2020.9.3On Education. Journal for Research and Debate. 39Hodgson, N. (2020). Post-critique, politics, and the political in educational philosophy. On Education. Journal for Research and Debate, 3(9). https://doi.org/10.17899/on_ed.2020.9.3

Her research focuses on the relationship between education, governance, and subjectivity. Her most recent publications include Manifesto for a Post-Critical Pedagogy. with Joris Vlieghe and Piotr Zamojski. Stefan Ramaekers and Kip KlinePalgraveAbout the Author Naomi Hodgson is Associate Professor of Education Studies at Liverpool Hope University ; Lewis University, US), titled Upbringing, Agency, and Subjectivity in an Algorithmic AgeCurrently she is working on a projectAbout the Author Naomi Hodgson is Associate Professor of Education Studies at Liverpool Hope University, UK. Her research focuses on the relationship between education, governance, and subjectivity. Her most recent publications include Manifesto for a Post- Critical Pedagogy, with Joris Vlieghe and Piotr Zamojski (Punctum Books, 2018) and Philosophical Presentations of Raising Children: The Grammar of Upbringing, with Stefan Ramaekers (Palgrave, 2019). Currently she is working on a project, with Stefan Ramaekers and Kip Kline (Lewis University, US), titled Upbringing, Agency, and Subjectivity in an Algorithmic Age.

#371), that is, in our very usage of words. His grammatical investigations seek "to understand something that is already in plain view" (#89), rather than a deeper, ultimate meaning. See Hodgson & Ramaekers (2019) for an articulation of. An alternative conception is found in Wittgenstein and the idea that. Essence is expressed by grammar. this in relation to the post-criticalAn alternative conception is found in Wittgenstein and the idea that "Essence is expressed by grammar" (1953, #371), that is, in our very usage of words. His grammatical investigations seek "to understand something that is already in plain view" (#89), rather than a deeper, ultimate meaning. See Hodgson & Ramaekers (2019) for an articulation of this in relation to the post-critical.