TY - JOUR
AU - Ehrhart‐Bornstein, Monika
AB - Abstract The neural crest‐derived adrenal medulla is closely related to the sympathetic nervous system; however, unlike neural tissue, it is characterized by high plasticity which suggests the involvement of stem cells. Here, we show that a defined pool of glia‐like nestin–expressing progenitor cells in the adult adrenal medulla contributes to this plasticity. These glia‐like cells have features of adrenomedullary sustentacular cells, are multipotent, and are able to differentiate into chromaffin cells and neurons. The adrenal is central to the body's response to stress making its proper adaptation critical to maintaining homeostasis. Our results from stress experiments in vivo show the activation and differentiation of these progenitors into new chromaffin cells. In summary, we demonstrate the involvement of a new glia‐like multipotent stem cell population in adrenal tissue adaptation. Our data also suggest the contribution of stem and progenitor cells in the adaptation of neuroendocrine tissue function in general. Stem Cells 2015;33:2037–2051 Nestin, Adult stem cells, Sustentacular cells, Stress, Plasticity, Neural stem cell‐like cells, Adrenal medulla, Lineage tracing Introduction Adult progenitor or stem cells are found in an increasing number of organs where they contribute to the renewal of organ‐specific cells. As a part of the sympathoadrenal system, the adrenal medulla develops from the neural crest, constituting a major lineage of neural crest derivates. Embryonic neural crest cells exhibit stem cell‐like characteristics, including self‐renewal and multipotency, which give rise to a wide variety of tissues. They become restricted in their potency during development as they migrate and reach their target tissues (reviewed in ref. [1]). It is now apparent, however, that many neural crest‐derived tissues appear to retain neural crest cells that are multipotent [2] or that even have pluripotent characteristics, such as the epidermal neural crest stem cells from the hair‐follicle [3]. A common neural crest progenitor for sympathetic neurons and chromaffin cells has been recently identified [4]; however, it is unclear whether multipotent neural crest cells persist in the adult adrenal medulla and contribute to the gland's adaptation to physiological needs. The adrenal medulla is part of the sympathetic nervous system, specifically the sympathoadrenomedullary system. The adrenal medulla is predominantly composed of chromaffin cells whose major secretory product is epinephrine. The endocrine system reacts to stress by activation of and interaction between the hypothalamic–pituitary–adrenal axis and the adrenomedullary neuroendocrine system, associated with secretion of adrenal hormones, adrenocortical glucocorticoids, and adrenomedullary epinephrine [5]. The proper adaptation of adrenomedullary function is, therefore, of fundamental importance to the body's stress response and regain of homeostasis. This adaptation can be achieved in different ways, such as adapting the levels of hormone secretion and their synthesis. This aspect has been extensively studied for the adrenal medulla [6-8]. The question of whether stem/progenitor cells persisting in the adult adrenal medulla contribute to the gland's adjustment and regeneration has, however, not been addressed. We recently showed that cells with progenitor properties can be enriched from the bovine [9] and human [10] adrenal medulla. In these in vitro studies, spheres from adrenal medulla, chromospheres, express progenitor markers including markers for neural stem cells, such as nestin, CD133, Notch1, nerve growth factor receptor, musashi1, and Snai2, and members of the SoxE subgroup, involved in migration and differentiation of neural crest derivates [11, 12], specifically into adrenal medulla [13]. Therefore, these previous studies suggest the persistence of neural crest‐derived progenitor cells in the adrenal medulla. However, these spheres were heterogeneous structures [9] and no lineage tracing was included in these studies. Furthermore, no in vivo studies examined the involvement of chromaffin stem/progenitor cells in the gland's adaptation to increased physiological needs. Our previous data indicate expression of the intermediate filament nestin by adrenomedullary progenitor cells. Nestin was initially identified as a marker of neural stem and progenitor cells [14, 15], but a wider distribution including its expression in different adult tissues is now generally accepted [16]. It is suggested that in adult tissues, nestin expression is most closely associated with a stem/progenitor cell population with multipotent properties and regenerative potential [16]. We therefore chose nestin as marker for the in vivo characterization of the stem/progenitor population in the adrenal medulla. This study shows in vivo in nestin–green fluorescent protein (GFP) mice, in which enhanced GFP (EGFP) is expressed under the regulation of the nestin regulatory elements [17], that nestin‐expressing cells are a cell population distinct from chromaffin cells with glia‐like features. We demonstrate in vitro the sphere forming capacity of these nestin–GFP expressing progenitors from the adrenal medulla. Furthermore, lineage tracing in nestin–CreER:Rosa26‐YFP mice showed the potential of these progenitors to differentiate to the endocrine, neural, and glial lineages. During repeated immobilization stress, a situation known in induce adrenal medulla activation, these cells are reduced in number. Lineage tracing reveals their in vivo differentiation into new chromaffin cells. Materials and Methods Animals Heterozygous nestin–GFP transgenic mice were generated as described previously [17] on the C57/BL6 genetic background. Both adults male and female nestin–GFP mice aged 2–5 months were used in this study. For lineage tracing studies, nestin–Cre‐ERT1mice [18] (Jackson Laboratories, Bar Harbor, ME, http://www.jax.org/; stock 012906) were bred with Rosa26‐YFP mice [19]. Ten days before the stress experiment, Cre expression was induced in adult Nes‐Cre‐ERT:Rosa26‐YFP mice by intraperitoneal injections of 100 µl tamoxifen (0.2 mg/ml in sunflower oil/10% ethanol) for 5 consecutive days. All mouse colonies were maintained under 12:12 hours light/dark cycle and fed ad libitum. Mice were killed by cervical dislocation after isoflurane anesthesia or, in the case of stress experiments, by CO2/O2 anesthesia. All animal experiments were approved according to the German Animal Welfare Act by the Landesdirektion Sachsen, Germany. Isolation and Culture of Nestin–GFP+ Cells from Adrenal Medulla Adrenals were explanted and placed in petri dishes with ice‐cold phosphate‐buffered saline (PBS). Fat tissue surrounding the adrenals and the adrenal cortex were carefully removed to isolate the medulla. All medullae were pooled, pelleted (1200 rpm, 6 minutes), and digested for 20 minutes at 37°C while shaking (1.8 mg/ml collagenase, 10 mg/ml BSA, 0.18 mg/ml DNAse in PBS; all from Sigma‐Aldrich, Saint Louis, MO, http://www.sigmaaldrich.com/). Digestion was stopped by washing twice in PBS; medullary cells were resuspended in 1 ml Dulbecco's modified Eagle medium (DMEM/F12)–high glucose (Gibco, Gran Island, NY, http://www.lifetechnologies.com/) containing 10% steroid‐free fetal bovine serum (charcoal/dextran treated; Hyclone Laboratories, South Logan, UT, www.hyclone.com), 1% antibiotic‐antimycotic solution (Gibco), 1% l‐Glutamine (PAA Laboratories, Cölbe, Germany, http://www3.gehealthcare.com/), and 20 ng/ml basic fibroblast growth factor (Sigma‐Aldrich). Isolated cells were cultured overnight in low attachment plates (Corning, Acton, MA, http://www.corning.com/lifesciences) [9, 20] at 37°C in a humidified atmosphere (95% O2, 5% CO2). After 24 hours, medium was changed to Neurobasal Medium (Gibco) with 2% B27 and 1% antibiotic‐antimycotic solution. After forming spheres (approximately 5 days), these were plated into two‐well chamber plates (ibidi, Planegg, Germany, http://ibidi.com/) coated with 1 mg/ml poly‐d‐lysine (Merck Millipore, Darmstadt, Germany, http://www.merckmillipore.com/)/3 µl/ml bFibronectin (R&D Systems, Minneapolis, MN, http://www.rndsystems.com/) to promote differentiation for 7 days. In long‐term experiments, differentiation of nestin–GFP cells was followed for 1 month. In Vitro EdU Staining Chromospheres were disrupted mechanically and treated with 10 mM 5‐ethynyl‐2′‐deoxy‐uridine (EdU, Invitrogen, Carlsbad, CA, http://www.lifetechnologies.com/) in culture medium for 24 hours. Cells were then plated into poly‐d‐Lysine/fibronectin coated two‐well chambers, as described above, for 3 hours and stained for EdU incorporation (Click‐iT EdU Alexa Fluor 594 or 555 Imaging Kit, Invitrogen). In Vivo EdU Incorporation Two protocols were followed to study proliferation of nestin–GFP progenitor cells in vivo and their behavior under stress conditions. In the first protocol, both the control and stress experimental groups of mice received one EdU injection (150 mg/kg; (Invitrogen) [21] on the first day of stress and were killed 24 hours later. In the second protocol, both groups received a daily injection of EdU (30 mg/kg; (Invitrogen) [22] to label all proliferating cells during a 7‐day period of immobilization. Mice were killed 24 hours after the last stress procedure; adrenals were explanted and processed as follows. Immunofluorescence Mice were anesthetized with isoflurane and killed by cervical dislocation. Adrenal glands were removed and fixed (4% paraformaldehyde (PFA), 4 hours), cryoprotected (30% PBS/sucrose, 4°C overnight), embedded in Tissue‐Tek Medium (OCT; Sakura Finetek, Torrance, CA, http://www.sakura.eu/), and stored at −80°C. Cryosections were cut at 11 µm (Leica CM 1900; Leica Biosystems, Wetzlar, Germany, http://www.leicabiosystems.com/) and mounted (Superfrost Plus slides; Thermo Scientific, Waltham, MA, http://www.thermoscientific.com). Cultured cells were fixed in 4% PFA in PBS for 15 minutes. Slides or cells were then immunostained using specific antibodies (Supporting Information Table S1) as detailed in Supporting Information. Immobilization Stress Adult male nestin–GFP mice (aged 2.5–4 months) were divided into control and experimental (stress) groups. Mice from the stress group were placed in individual cages whereas control mice were not disturbed. Two days later, restraining (2 hours) was initiated for 6 consecutive days at 9.00 am using mouse restrainers (Braintree Scientific, Braintree, MA, http://www.braintreesci.com/). Mice were weighed on the first day of preadaptation, the first and the last day of the stress experiments (n = 19). For time course experiments, mice were killed on the 1st, 3rd, or 6th day of the stress experiments (n = 3 in each group). Adrenals were collected, weighed, and processed for immunostaining. To study adrenomedullary nestin–GFP positive cells during recovery following prolonged stress, an additional protocol was applied which included a resting period of 7 days after the stress procedure. Mice were killed on the last day of the period. Nestin–CreER:Rosa26‐YFP mice were subjected to the same restraining protocol and killed and studied after 6 days of immobilization stress. Results Nestin–GFP Positive Cells Are Present in Adult Mouse Adrenal Medulla By using the nestin–GFP transgenic mouse model, which has been validated in several tissues to label stem and progenitor cells [17, 23-28], we aimed to identify the population of progenitor cells in the adult adrenal medulla and elucidate their role in physiologically active processes such as stress. Nestin is a type IV intermediate filament protein expressed in multipotent neural stem cells [15]; it has been widely accepted as a progenitor marker in the developing nervous system and some neurogenic and proliferative areas in the adult brain. Within the adrenal medulla, the number of nestin–GFP positive cells was approximately 6%. They displayed a pyramidal shape with one to three processes or a spherical shape with almost no extensions (Fig. 1A–1E). Open in new tabDownload slide Nestin–green fluorescent protein (GFP) expressing cells in the adrenal medulla. (A): Overview of the adrenal medulla from a nestin–GFP transgenic adult mouse showing the distribution of nestin–GFP progenitor cells. Scale bar = 200 µm (Supporting Information Movie S1). (B–E): Nestin–GFP‐positive cells within the adrenal medulla display different morphologies: elongated with one process (B), with geometric shape with one long basal process (C), rounded (D), and with several processes (E). (F): Immunofluorescent staining for CD31 of adrenal medulla from nestin–GFP mouse to delimit blood vessels (BV). Nestin–GFP‐positive cells extend one of their processes towards the BV. Scale bar = 50 µm. (G): Higher magnification of nestin–GFP cell extending its long process towards the BV. Scale bar = 50 µm. Open in new tabDownload slide Nestin–green fluorescent protein (GFP) expressing cells in the adrenal medulla. (A): Overview of the adrenal medulla from a nestin–GFP transgenic adult mouse showing the distribution of nestin–GFP progenitor cells. Scale bar = 200 µm (Supporting Information Movie S1). (B–E): Nestin–GFP‐positive cells within the adrenal medulla display different morphologies: elongated with one process (B), with geometric shape with one long basal process (C), rounded (D), and with several processes (E). (F): Immunofluorescent staining for CD31 of adrenal medulla from nestin–GFP mouse to delimit blood vessels (BV). Nestin–GFP‐positive cells extend one of their processes towards the BV. Scale bar = 50 µm. (G): Higher magnification of nestin–GFP cell extending its long process towards the BV. Scale bar = 50 µm. The adrenal medulla is a highly vascularized organ and we found nestin–GFP positive cells to be in close contact with blood vessels (Fig. 1F, 1G). Blood‐derived factors might thus influence and regulate their physiological behavior. To further characterize potential subpopulations and the commitment of these nestin–GFP progenitors, we immunostained the adrenal medulla for markers of the chromaffin, glia and neural lineages. Tyrosine hydroxylase (TH), the rate‐limiting enzyme of catecholamine synthesis, is expressed by adrenomedullary chromaffin cells and thus delimits the adrenal medulla from the cortex (Supporting Information Fig. S1). No cellular colocalization of TH and nestin–GFP was detected in any adult adrenal studied, suggesting that nestin is not expressed by differentiated or mature chromaffin cells (Fig. 2A; Supporting Information Fig. S1, Video 1). Another adrenomedullary protein is chromogranin A (CgA), the major protein stored and released by the large dense cored vesicles characteristic for chromaffin cells [29]. Alike immunostaining for TH staining for CgA did not colocalize with GFP (Fig. 2B). Open in new tabDownload slide Subpopulations of the Nestin–green fluorescent protein (GFP) Cell Population within Adrenal Medulla. (A, B) Immunofluorescent staining revealed that nestin–GFP cells in the adrenal medulla are a cell population distinct from chromaffin cells; GFP expression (white arrows) did not overlap with markers for differentiated chromaffin cells such as tyrosine hydroxylase (A) and chromogranin A (B). (C): The majority of the nestin–GFP‐positive cells did not coexpress the neuronal marker β‐III tubulin (Supporting Information Fig. S3). (D, E) Nestin–GFP‐positive cells were found to express glia markers (yellow arrows). (D): Of the nestin–GFP‐positive cells, 62% were costained for glial fibrillary acidic protein (GFAP) (1037 double positive nestin–GFP/GFAP out of 1683 nestin–GFP positive from 26023 DAPI nuclei counted; n = 9). (E): All nestin–GFP‐positive cells were costained for S100b; 72% of those showed staining for S100b protein in their whole cell body (1104 double positive nestin–GFP/S100b out of 1528 nestin–GFP positive from 26003 DAPI nuclei counted; n = 9) (Supporting Information Movie S2). (F): All nestin–GFP‐positive cells were coexpressing BLBP marker for Schwann cell precursors of the peripheral nervous system. (G): Of the nestin–GFP‐positive cells, 59% expressed the transcription factor Sox10, crucial in regulating neural crest stem cell development and differentiation. Scale bar = 50 µm. Abbreviations: BLBP, brain lipid‐binding protein; DAPI, 4′,6‐diamidino‐2‐phenylindole; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; TH, tyrosine hydroxylase. Open in new tabDownload slide Subpopulations of the Nestin–green fluorescent protein (GFP) Cell Population within Adrenal Medulla. (A, B) Immunofluorescent staining revealed that nestin–GFP cells in the adrenal medulla are a cell population distinct from chromaffin cells; GFP expression (white arrows) did not overlap with markers for differentiated chromaffin cells such as tyrosine hydroxylase (A) and chromogranin A (B). (C): The majority of the nestin–GFP‐positive cells did not coexpress the neuronal marker β‐III tubulin (Supporting Information Fig. S3). (D, E) Nestin–GFP‐positive cells were found to express glia markers (yellow arrows). (D): Of the nestin–GFP‐positive cells, 62% were costained for glial fibrillary acidic protein (GFAP) (1037 double positive nestin–GFP/GFAP out of 1683 nestin–GFP positive from 26023 DAPI nuclei counted; n = 9). (E): All nestin–GFP‐positive cells were costained for S100b; 72% of those showed staining for S100b protein in their whole cell body (1104 double positive nestin–GFP/S100b out of 1528 nestin–GFP positive from 26003 DAPI nuclei counted; n = 9) (Supporting Information Movie S2). (F): All nestin–GFP‐positive cells were coexpressing BLBP marker for Schwann cell precursors of the peripheral nervous system. (G): Of the nestin–GFP‐positive cells, 59% expressed the transcription factor Sox10, crucial in regulating neural crest stem cell development and differentiation. Scale bar = 50 µm. Abbreviations: BLBP, brain lipid‐binding protein; DAPI, 4′,6‐diamidino‐2‐phenylindole; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; TH, tyrosine hydroxylase. Immunostaining for β‐III tubulin (Tuj1), an intermediate filament commonly expressed in early and differentiated neurons [30], was also located in the adrenal medulla (Fig. 2C). These Tuj1‐positive neurons with labelled postganglionic processes were intermingled with the chromaffin cells. Most of these cells were negative for GFP with processes often running parallel to nestin–GFP positive processes (Supporting Information Fig. S2A). However, some nestin–GFP positive cells coexpressed β‐III tubulin; these were characterized by a weaker GFP signal (dim cells) compared with the neighbouring nestin–GFP+/Tuj1‐ cells (Supporting Information Fig. S2B–S2E), suggesting downregulation of nestin–GFP expression with differentiation [31]. This is similar to the brain, where the morphology of these progenitors and the brightness of the GFP signal are indicative of the level of the cells' commitment [17]. The derivation of neurons and chromaffin cells from nestin–GFP expressing progenitor cells are in accordance with the recent in vivo finding that sympathetic neurons and chromaffin cells share a common progenitor in the neural crest [4]. Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed by cells of astroglial lineage in the central nervous system (CNS) [32]. However, neural stem cells (NSC) in the adult brain have also been shown to have characteristics of astrocytes including the expression of GFAP [33]. By immunostaining for GFAP (Fig. 2D) we found that approximately 62% of the nestin–GFP positive cell population within the adrenal medulla was costained for GFAP, suggesting similar features of these progenitor cells with NSC. In addition to chromaffin and neuronal cells, the adrenal medulla contains supportive sustentacular cells with morphologic, functional, and antigenic properties similar to Schwann and satellite cells [34]. S100b is a calcium binding protein expressed by sustentacular cells of the adult human adrenal [34, 35] and the adrenal medulla of a wide range of mammals [36]. We found that all nestin–GFP positive cells in the adrenal medulla expressed the astrocyte marker S100b to a certain extent; 72% of nestin–GFP positive cells costained for S100b in their cell body and processes (Fig. 2E; Supporting Information Video 2). The remaining 28% of nestin–GFP positive cells showed weaker immunostaining for S100b in the processes only. Vice versa, all S100b positive cells were found to coexpress nestin–GFP (Supporting Information Video 2). The adrenal medulla is closely related to sympathetic ganglia; both contain derivates of the sympathoadrenal lineage from the neural crest. Similar to the adrenal medulla, fetal and postnatal sympathetic ganglia also contain nestin–expressing progenitor cells [37]. In contrast, however, nestin expression in sympathetic ganglia was undetectable in adult ganglia after the cells acquired S100 expression [37]. The coexpression of nestin and S100 in the adrenomedullary sustentacular cells indicates that, in contrast to sympathetic ganglia, the cells are not fully differentiated. Further characterization of this cell population included immunostaining for brain lipid‐binding protein (BLBP), a marker for Schwann cell precursors of the peripheral nervous system [38]. All nestin–GFP cells expressed BLBP (Fig. 2F), which is similar to postnatal sympathetic ganglia [37] where nestin+ progenitor cells coexpressed BLBP, defining a population of progenitors with both glial and neuronal progenitor characteristics. Furthermore, a subpopulation of nestin–GFP+ cells (59% of the nestin–GFP+ cells) coexpressed Sox10 (Fig. 2G). The transcription factor Sox10 is crucial in regulating neural crest stem cell development and differentiation (reviewed in ref. [39]) Sox10 is involved in Schwann cell differentiation but also preserves the neuronal potential of neural crest‐derived stem cells [12]. Moreover, Sox 10 is expressed by migrating neural crest‐derived sympathoadrenal stem cells and its expression is required for the formation of the adrenal medulla [13]. The expression of Sox10 by sympathoadrenal progenitor cells in situ in the adrenal medulla is in accordance with our previous data from the human adrenal medulla showing the expression of Sox10 by in vitro enriched progenitor cells in sphere culture [10]. Thus, these data further support the neural crest‐like properties of the sympathoadrenal progenitor cells in the adult adrenal medulla. Together, these results imply that nestin–GFP positive cells located in the adrenal medulla represent a population distinct from that of mature chromaffin cells. Adrenal medulla and sympathetic ganglia are similar in many respects; however, some significant differences exist between the two tissues, including life‐long proliferation of adrenomedullary cells and the adaptation to physiological needs which seems to include the persistence of differentiation competent progenitor cells in the adult. These cells correspond to a glia‐like population of cells, the sustentacular cells of the adrenal medulla. Similar to radial glia‐like cells in the adult brain [21, 40], they might have stem cell features and the potential to differentiate to the neural lineages, activated when required depending on physiological needs. This correlates with the observation that the nestin–GFP brightness seems to depend on the level of the cells' commitment; for example, that some of the dim cells coexpressed β‐III tubulin. Nestin–GFP Cells In Vitro Form Spheres, Proliferate, and Have Neurogenic Potential To characterize nestin–GFP cells in vitro in culture, the entire adrenal cortex was carefully removed to obtain a pure adrenal medullary culture. Isolated cells from the adrenals of nestin–GFP mice were cultured in sphere‐forming conditions in low‐attachment plates where they formed chromospheres the day after isolation. This is similar to what has been described for nestin–GFP positive cells from other tissues from the same mouse model [17, 23-27]. After 24 hours in culture all nestin–GFP positive cells were included in spheres which were heterogeneous concerning the GFP‐expression. During 7 days of culture in low attachment conditions, GFP+ cells in these spheres proliferated resulting in a considerably increased nestin–GFP population within the spheres (Fig. 3A; Supporting Information Fig. S3A). To study the differentiation potential of chromosphere cells, spheres were mechanically disrupted and cells then seeded into poly‐d‐lysine/fibronectin coated plates and observed for 1 month. Nestin–GFP positive cells further proliferated and migrated out of the spheres (Fig. 3A; Supporting Information Video 3) and acquired a neuron‐like morphology (Supporting Information Fig. S3B). This is in accordance with the expression of β‐III tubulin (Tuj1) by these neuron‐like cells in vitro (Fig. 4A; Supporting Information Fig. S3B, S3C). It has been shown that GFAP positive NSCs are able to form multipotent spheres [41]. Accordingly, GFAP (Fig. 4B) was coexpressed with nestin–GFP within the chromospheres while more differentiated cells were found to migrate from the spheres and to differentiate, expressing S100b (Fig. 4C). Furthermore, the proliferative potential of these cells was proven by EdU incorporation after disruption of primary spheres (see below). Open in new tabDownload slide Nestin–green fluorescent protein (GFP) Cells in vitro Form Spheres and Proliferate. (A): Small spheres (chromospheres) with one or two GFP‐positive cells were observed the day after cell isolation. GFP‐positive cells within the spheres proliferated under low‐attachment conditions for 1 week when the spheres were plated into poly‐d‐Lysine coated plates and cultured for up to 1 month. Nestin–GFP cells continued to divide and proliferate under adherent conditions (n = 3 experiments, 15 mice each). Scale bar = 1 day 50 µm; 7 days 100 µm; 14 days and 1 month 200 µm (Supporting Information Figs. S4–S6). (B): The 24‐hour treatment with EdU and mechanical disruption of spheres (2–3 days after isolation) revealed the proliferation and self‐renewal capacity of nestin–GFP cells in vitro. Arrows indicate nestin–GFP cells that incorporated EdU (n = 5 experiments, 15mice each) (Supporting Information Movie S3). Scale bar = 50 µm. Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; EdU, 5‐Ethynyl‐2′‐deoxy‐uridine; GFP, green fluorescent protein. Open in new tabDownload slide Nestin–green fluorescent protein (GFP) Cells in vitro Form Spheres and Proliferate. (A): Small spheres (chromospheres) with one or two GFP‐positive cells were observed the day after cell isolation. GFP‐positive cells within the spheres proliferated under low‐attachment conditions for 1 week when the spheres were plated into poly‐d‐Lysine coated plates and cultured for up to 1 month. Nestin–GFP cells continued to divide and proliferate under adherent conditions (n = 3 experiments, 15 mice each). Scale bar = 1 day 50 µm; 7 days 100 µm; 14 days and 1 month 200 µm (Supporting Information Figs. S4–S6). (B): The 24‐hour treatment with EdU and mechanical disruption of spheres (2–3 days after isolation) revealed the proliferation and self‐renewal capacity of nestin–GFP cells in vitro. Arrows indicate nestin–GFP cells that incorporated EdU (n = 5 experiments, 15mice each) (Supporting Information Movie S3). Scale bar = 50 µm. Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; EdU, 5‐Ethynyl‐2′‐deoxy‐uridine; GFP, green fluorescent protein. Open in new tabDownload slide In vitro Differentiation of Nestin–green fluorescent protein (GFP) Cells. Nestin–GFP cells after being plated into a poly‐d‐Lysine coated plates, started to differentiate (Supporting Information Movie S3). Three different cell populations were characterized by immunofluorescence: (A): Cells, coexpressing the early neuronal marker β‐III tubulin and GFP. Note the weaker GFP expression in these cells as compared with the cells in (B) and (C) indicating the downregulation of nestin. (B): Nestin–GFP‐positive cells coexpressing glial fibrillary acidic protein and (C) S100b with glia‐like morphology. Scale bar = 50 µm (Supporting Information Fig. S4). Abbreviations: GFP, green fluorescent protein; DAPI, 4′,6‐diamidino‐2‐phenylindole. Open in new tabDownload slide In vitro Differentiation of Nestin–green fluorescent protein (GFP) Cells. Nestin–GFP cells after being plated into a poly‐d‐Lysine coated plates, started to differentiate (Supporting Information Movie S3). Three different cell populations were characterized by immunofluorescence: (A): Cells, coexpressing the early neuronal marker β‐III tubulin and GFP. Note the weaker GFP expression in these cells as compared with the cells in (B) and (C) indicating the downregulation of nestin. (B): Nestin–GFP‐positive cells coexpressing glial fibrillary acidic protein and (C) S100b with glia‐like morphology. Scale bar = 50 µm (Supporting Information Fig. S4). Abbreviations: GFP, green fluorescent protein; DAPI, 4′,6‐diamidino‐2‐phenylindole. To study the proliferative capacity of nestin–GFP positive cells in vitro, we treated the cells with EdU 24 hours after disruption of primary spheres and seeding the cells for 3 hours into coated plates (Fig. 3B). We found that up to 66.43% of the EdU positive cells in the spheres were also positive for nestin–GFP and 40.5% of the nestin–GFP+ cells were also positive for EdU (n = 5). This result reveals that in vitro most of the proliferative cells within the chromospheres correspond to the nestin–GFP positive population. This supports the in vitro observation (Figs. 3A; Supporting Information Fig. S3A) of the proliferative capacity of nestin–GFP+ cells described above which resulted in highly homogenous GFP+ spheres. In vivo, only approximately 1% of the GFP+ cells incorporated EdU under basal conditions (Fig. 6J; Supporting Information Fig. S6B, data from control groups). Thus, the proliferative activity of the nestin–GFP cells was increased by the in vitro conditions. As Prominin‐1 (CD133) is a frequently used marker of multipotent NSC with self‐renewal capacity [42], we examined whether CD133 is expressed by nestin–GFP+ cells from the adrenal medulla. Flow cytometry revealed a population of adrenomedullary cells which expressed CD133 (Supporting Information Fig. S4), adding another feature shared with NSC. We identified three different subpopulations of CD133+ cells when sorting the cells for nestin–GFP intensity: nestin–GFP negative, medium and bright cells (Supporting Information Fig. S4A). We then analyzed Prominin‐1 expression in these subpopulations and found it to correlate with GFP intensity. While 24% of the nestin–GFP negative group and 27.4% of the medium nestin–GFP cells were Prominin‐1 positive (Supporting Information Fig. S4B, S4C, S4E), 73.6% of the cells with high intensity of nestin–GFP coexpressed Prominin‐1 (Supporting Information Fig. S4D, S4E). These in vitro findings suggest that nestin–GFP positive cells share properties with NSC: they are able to form spheres and proliferate in low‐attachment conditions giving rise to highly homogeneous nestin–GFP positive spheres. In addition, in vitro culture revealed coexpression of S100b and GFAP as well as Prominin‐1 with nestin–GFP. Furthermore, GFP‐positive cells in adherent pro‐neural conditions differentiated giving rise to neuron‐like cells characterized by β‐III tubulin expression. Nestin Expressing Progenitor Cells Within Adrenal Medulla Are Multipotent The nestin–GFP mouse model is a valuable tool for studying progenitor cells in the adrenal medulla. However, since GFP fluorescence declines with the cells' differentiation, we bred an inducible mouse model, nestin–CreER:Rosa26‐YFP, to trace the progeny of the nestin expressing progenitor cells within the adrenal medulla. For in vitro lineage tracing, adrenomedullary cells were isolated following the procedure described for the nestin–GFP mouse. Induction of YFP expression via 4‐hydroxy‐tamoxifen was performed directly on the day of isolation to study the multipotency of the nestin expressing progenitors and to trace all the lineages that the nestin progenitors are able to give rise to (Fig. 5). After induction, secondary chromospheres were plated to promote differentiation of the cells. Progenitor cells from the adrenal medulla gave rise to three different populations: 35.98% neuron‐like cells (YFP+/β‐III tubulin+ cells), 36.21% glia (YFP+/S100b+), and 26.41% chromaffin cells (YFP+/CgA+) (n = 3) (Fig. 5A, 5B). Together, in vitro lineage tracing proves that nestin‐expressing progenitors in the adrenal medulla are able to differentiate into the three adrenomedullary lineages, chromaffin, neural, and glia. Open in new tabDownload slide In vitro Lineage tracing of nestin‐expressing progenitors from NestinCreERT2:R26‐YFP mice. (A): Immunofluorescent staining of in vitro differentiated cells from the adrenal medulla of NestinCreERT2:R26‐YFP adult mice show that nestin progenitors are multipotent in vitro. YFP‐positive cells were immunostained for glia (S100b), neuronal (β‐III tubulin), and chromaffin (chromogranin A [CgA]). Scale bar = 50µm. (B): nestin–YFP cells from CreERT2/R26YFP mice in vitro give rise to chromaffin (102 ± 1.31 CgA+/YFP+ double positive cells out of 314 ± 3.89 YFP+ cells from 4967 ± 40.80 DAPI nuclei, n = 3), neuronal (172 ±1.38 β‐III tubulin+/YFP+ double positive cells out of 479 ± 2.99 YFP+ cells from 8352 ± 51.01 DAPI nuclei, n = 3), and glia (151 ± 3.84 S100b+/YFP+ double positive cells out of 417 ± 4.87 YFP+ cells from 4886 ± 43.78 DAPI nuclei, n = 3) cells. Abbreviations: CgA, chromogranin A; DAPI, 4′,6‐diamidino‐2‐phenylindole. Open in new tabDownload slide In vitro Lineage tracing of nestin‐expressing progenitors from NestinCreERT2:R26‐YFP mice. (A): Immunofluorescent staining of in vitro differentiated cells from the adrenal medulla of NestinCreERT2:R26‐YFP adult mice show that nestin progenitors are multipotent in vitro. YFP‐positive cells were immunostained for glia (S100b), neuronal (β‐III tubulin), and chromaffin (chromogranin A [CgA]). Scale bar = 50µm. (B): nestin–YFP cells from CreERT2/R26YFP mice in vitro give rise to chromaffin (102 ± 1.31 CgA+/YFP+ double positive cells out of 314 ± 3.89 YFP+ cells from 4967 ± 40.80 DAPI nuclei, n = 3), neuronal (172 ±1.38 β‐III tubulin+/YFP+ double positive cells out of 479 ± 2.99 YFP+ cells from 8352 ± 51.01 DAPI nuclei, n = 3), and glia (151 ± 3.84 S100b+/YFP+ double positive cells out of 417 ± 4.87 YFP+ cells from 4886 ± 43.78 DAPI nuclei, n = 3) cells. Abbreviations: CgA, chromogranin A; DAPI, 4′,6‐diamidino‐2‐phenylindole. Stress Promotes Differentiation of Nestin–GFP‐Positive Cells Within the Adrenal Medulla Numerous studies have addressed the involvement of the adrenal medulla in response to different kinds of stressors [8, 43]. However, the potential role of progenitor cells in adrenal medulla has not yet been studied. In this study, nestin–GFP mice were submitted to repeated immobilization stress (2 hours for 6 consecutive days) (Fig. 6A). Before stress, mice were isolated in different cages, while control mice were kept together in the same cages (n = 4 mice per group and n = 5 experiments) with access to food and water ad libitum. In contrast to control mice, restrained mice lost significant body weight (Fig. 6B), while their adrenals were increased in size by approximately 22% (Fig. 6C). Proliferating cells were identified following two protocols in different sets of experiments; first by one EdU injection (150 mg/kg) [21] at the first day of restraint (Fig. 6J, 6K) and second with one daily injection of EdU (30 mg/kg) over the entire stress period (Supporting Information Fig. S5). The first protocol resulted in a significant increase in the proliferation rate of nestin–GFP cells at day 1 (3.68 ± 1.04% EdU+ nestin–GFP+ out of total nestin–GFP+ cells) compared with unstressed controls (0.68 ± 0.62% EdU+ nestin–GFP+ out of total nestin–GFP+ cells). This result suggests that nestin–GFP positive cells proliferate at a low rate at basal conditions and react with increased proliferation to highly demanding situations, such as one day of stress. The multi‐injection experiment allowed study of the proliferative capacity of nestin–GFP positive cells when stress became chronic. While in unstressed controls, the number of EdU‐positive cells was increased compared with the single EdU injection (1.55 ± 0.27% EdU+/nestin–GFP+ out of total nestin–GFP+ cells) and the number of EdU+/nestin–GFP+ cells was unchanged after chronic stress (2.89 ± 0.38% EdU+/nestin–GFP+ out of total nestin–GFP+ cells). This could be due to the fact that the GFP signal is downregulated when the cells differentiate. Open in new tabDownload slide Restraint Stress Affects the Population of Progenitor Cells. (A): Repeated restraint stress of nestin–green fluorescent protein (GFP) mice, 2 hours for 6 consecutive days, was performed to identify the involvement of nestin‐progenitors in the gland's adaptation to stress. (B): Body weight of the animals was affected with a significant difference in Δ weight between control and stressed mice. (C): As a consequence of the stress also adrenal gland weight is significantly affected, with enlarged adrenals in the stressed animals (A–C: n = 18 per experimental group). (D): After 6 days of stress, the number of nestin–GFP cells in the adrenal medulla was significantly reduced from 8.32 ± 1.36 in control mice to 4.16 ± 0.90%GFP+ cells (n = 14). (E): Immunofluorescent staining of adrenal medulla revealed differences in cell numbers between controls and stressed mice concerning cells double positive for nestin–GFP/glial fibrillary acidic protein (GFAP) or S100b. Both cell populations were reduced in stressed mice after 6 days of repeated immobilization. Scale bar = 200µm. (F‐I): Time course experiments demonstrated time dependent differences in the effect of stress on the different nestin–GFP subpopulations. Animals were killed at days 1, 3, and 7, after 1, 3, or 6 2‐hour periods of immobilization stress, respectively. (n = 3 per experimental group and time points). (G): The number of GFP‐positive cells was significantly reduced at day seven after 6 days of stress, while no differences were observed at days 1 and 3 (Supporting Information Table S2). (H): The number of GFAP/ nestin–GFP double positive cells was already reduced after the first stress period and remained reduced in stressed mice in comparison with controls (Supporting Information Table S2). (I): In contrast, cells double positive for S100b/ nestin–GFP were increased in the stressed group at day 1 but significantly decreased after 6 days of repeated stress. (J): One injection of 150 mg/kg EdU was given to nestin–GFP mice the first day of stress before start the restrain. Double positive cells (EdU+ nestin–GFP+) were found to be significantly increased in stressed animals by day one. (n = 4 in stress group and n = 3 in control group). (K): Detail of two neighboring EdU positive cells where one is positive for nestin–GFP and the other is negative. (L): To study the behavior of nestin–GFP‐positive cells after stress, mice were allowed to rest for a period of 7 days. (n = 4 in stress group and n = 3 in control group). (LL): Body weight of control mice was maintained over the experimental period and one week after. Stressed mice had lost weight after 7 days of stress; after 1 week of resting (day 14) they had almost recovered their original weight. (M): Control and stressed mice after 7 days resting period showed no significant differences in percentage of nestin–GFP‐positive cells normalize with DAPI nuclei. (D, G, H, I, M): Percentage GFP positive cells are expressed normalized to DAPI‐stained nuclei. (J): Percentage double positive cells (EdU+ nestin–GFP+) are expressed normalized to total number of nestin–GFP‐positive cells. Statistical significance was determined using Student's t test. Numbers are expressed as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; EdU, 5‐ethynyl‐2′‐deoxy‐uridine; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein. Open in new tabDownload slide Restraint Stress Affects the Population of Progenitor Cells. (A): Repeated restraint stress of nestin–green fluorescent protein (GFP) mice, 2 hours for 6 consecutive days, was performed to identify the involvement of nestin‐progenitors in the gland's adaptation to stress. (B): Body weight of the animals was affected with a significant difference in Δ weight between control and stressed mice. (C): As a consequence of the stress also adrenal gland weight is significantly affected, with enlarged adrenals in the stressed animals (A–C: n = 18 per experimental group). (D): After 6 days of stress, the number of nestin–GFP cells in the adrenal medulla was significantly reduced from 8.32 ± 1.36 in control mice to 4.16 ± 0.90%GFP+ cells (n = 14). (E): Immunofluorescent staining of adrenal medulla revealed differences in cell numbers between controls and stressed mice concerning cells double positive for nestin–GFP/glial fibrillary acidic protein (GFAP) or S100b. Both cell populations were reduced in stressed mice after 6 days of repeated immobilization. Scale bar = 200µm. (F‐I): Time course experiments demonstrated time dependent differences in the effect of stress on the different nestin–GFP subpopulations. Animals were killed at days 1, 3, and 7, after 1, 3, or 6 2‐hour periods of immobilization stress, respectively. (n = 3 per experimental group and time points). (G): The number of GFP‐positive cells was significantly reduced at day seven after 6 days of stress, while no differences were observed at days 1 and 3 (Supporting Information Table S2). (H): The number of GFAP/ nestin–GFP double positive cells was already reduced after the first stress period and remained reduced in stressed mice in comparison with controls (Supporting Information Table S2). (I): In contrast, cells double positive for S100b/ nestin–GFP were increased in the stressed group at day 1 but significantly decreased after 6 days of repeated stress. (J): One injection of 150 mg/kg EdU was given to nestin–GFP mice the first day of stress before start the restrain. Double positive cells (EdU+ nestin–GFP+) were found to be significantly increased in stressed animals by day one. (n = 4 in stress group and n = 3 in control group). (K): Detail of two neighboring EdU positive cells where one is positive for nestin–GFP and the other is negative. (L): To study the behavior of nestin–GFP‐positive cells after stress, mice were allowed to rest for a period of 7 days. (n = 4 in stress group and n = 3 in control group). (LL): Body weight of control mice was maintained over the experimental period and one week after. Stressed mice had lost weight after 7 days of stress; after 1 week of resting (day 14) they had almost recovered their original weight. (M): Control and stressed mice after 7 days resting period showed no significant differences in percentage of nestin–GFP‐positive cells normalize with DAPI nuclei. (D, G, H, I, M): Percentage GFP positive cells are expressed normalized to DAPI‐stained nuclei. (J): Percentage double positive cells (EdU+ nestin–GFP+) are expressed normalized to total number of nestin–GFP‐positive cells. Statistical significance was determined using Student's t test. Numbers are expressed as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; EdU, 5‐ethynyl‐2′‐deoxy‐uridine; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein. In accordance, the number of nestin–GFP progenitor cells in the adrenal medullae of stressed mice was significantly reduced (by approximately 50%), suggesting their involvement in the gland's adaptation (Fig. 6D, 6E). When the mice were allowed to recover for 7 days following the restrained stress (Fig. 6L) they regained their body weight (Fig. 6LL). After this resting period, the number of nestin–GFP positive cells had returned to the levels of those of unstressed mice (Fig. 6M). Time course experiments (Fig. 6F) revealed no difference in the number of nestin–GFP positive cells between control and stressed mice at days 1 and 3 of immobilization stress (Fig. 6G), while after 7 days (chronic stress) the number of GFP positive cells was significantly decreased (Fig. 6D, 6E, 6G). To assess different subpopulations of nestin–GFP+ cells, adrenals were immunostained for GFAP and S100b. We found that GFAP+/ nestin–GFP+ cells were already significantly reduced at the first day of stress and also at days three and seven compared with control mice (Fig. 6H; Supporting Information Table S2). Contrarily, the number of GFP+ cells costained for S100b was increased at the first day of stress and decreased after 7 days in comparison with control mice (Fig. 6I; Supporting Information Table S2). These results, with a decline of GFAP+/ nestin–GFP+ cells and a significant increase of S100b+/ nestin–GFP+ cells after one day of immobilization stress suggest that nestin–GFP progenitors in the adrenal medulla behave as activated glia cells during shorter periods of stress and that they proliferate to increase this glia population as demonstrated by increased EdU incorporation. These sustentacular cells might be important in maintaining the highly activated chromaffin cells during these short periods. But when the stress becomes chronic, this supportive glial differentiation might not be sufficient to cope with the requirements leading to a further reduction in the nestin–GFP cells. Lineage Tracing of Nestin Progenitors Under Stress Conditions While the nestin–GFP mouse model proved the involvement of nestin–GFP expressing progenitors in stress adaption, the nestin–CreER:Rosa26‐YFP mouse provides the possibility for lineage tracing and identification of the physiological role of these cells in this highly demanding biological situation. In these animals, YFP expression is induced upon tamoxifen administration and activation of Cre in a proportion of nestin positive cells, promoting the permanent expression of YFP in those cells and their progeny. (Tamoxifen‐induced recombination probably is effective only in a fraction of potential target cells; therefore, the overall contribution of adult stem cells using this approach is not quantitative.) The in vivo validation of the nestin–CreER:Rosa26‐YFP mouse revealed the efficiency of Cre recombination by 100 µl of tamoxifen (0.2 mg/ml in sunflower oil/10% ethanol) injections for 5 consecutive days in this mouse model. Two weeks post induction, tamoxifen‐induced YFP expression was observed in cells of the three lineages identified in vitro, specifically cells of the neuronal, glia, and chromaffin lineages as identified by immunohistochemistry (Fig. 7A). Open in new tabDownload slide In vivo Fate of adrenal medulla nestin progenitor cells under normal and stress conditions. (A): Immunoflourescence of NestinCreERT2:R26YFP adult adrenal glands 2 weeks after tamoxifen induction of Cre‐recombination reveals in vivo YFP+ cells coexpressing markers for chromaffin (chromogranin A [CgA]), glia (S100b), and neuronal (β‐III tubulin) lineages (yellow arrows) proving the differentiation of nestin progenitors in the three major cell types of the adrenal medulla. Scale bar = 50µm. (B): After tamoxifen induction (5 consecutive days) and 10 days resting time the identical stress protocol was applied to the NestinCreERT2:R26YFP as for the Nestin–green fluorescent protein (GFP) mice (n = 6 mice per experimental group). (C): After 6 days of stress, the number of YFP+ cells was significantly increased compared with unstressed animals (127.67 YFP+ cells in controls and 296 YFP+ cells in stressed mice; cells were counted on five slices from the complete adrenal medulla, total number of cells, n = 6). (D): This increase was due to a significant increase in YFP‐positive chromaffin cells characterized by immunostaining for CgA (in controls 43.17 cells were double positive for YFP+/CgA+ in comparison with stressed mice where 113.5 cells were double positive for YFP+/CgA+, total number of cells). Populations of YFP+/S100+ (glia) and neuronal (YFP+/β‐III tubulin+) cells was similar in both experimental groups (Supporting Information Fig. S6). (C‐D): Numbers are expressed as mean ± SD. Statistical significance was determined using Student's t test.; *, p < 0.05. (E): In summary, under basal conditions the glia‐like, nestin–GFP expressing stem cells express the progenitor markers nestin and Prominin‐1, as well as the glia markers glial fibrillary acidic protein and S100b. These cells are almost quiescent under basal conditions. Under stress conditions, immobilization stress in this case, a fraction of the nestin–GFP expressing glia‐like progenitors proliferate and differentiate towards the specific cells required to cope with stress, predominantly into the chromaffin lineage; also differentiation into neuron‐like cells was observed. Abbreviations: CgA, chromogranin A; GFAP, glial fibrillary acidic protein. Open in new tabDownload slide In vivo Fate of adrenal medulla nestin progenitor cells under normal and stress conditions. (A): Immunoflourescence of NestinCreERT2:R26YFP adult adrenal glands 2 weeks after tamoxifen induction of Cre‐recombination reveals in vivo YFP+ cells coexpressing markers for chromaffin (chromogranin A [CgA]), glia (S100b), and neuronal (β‐III tubulin) lineages (yellow arrows) proving the differentiation of nestin progenitors in the three major cell types of the adrenal medulla. Scale bar = 50µm. (B): After tamoxifen induction (5 consecutive days) and 10 days resting time the identical stress protocol was applied to the NestinCreERT2:R26YFP as for the Nestin–green fluorescent protein (GFP) mice (n = 6 mice per experimental group). (C): After 6 days of stress, the number of YFP+ cells was significantly increased compared with unstressed animals (127.67 YFP+ cells in controls and 296 YFP+ cells in stressed mice; cells were counted on five slices from the complete adrenal medulla, total number of cells, n = 6). (D): This increase was due to a significant increase in YFP‐positive chromaffin cells characterized by immunostaining for CgA (in controls 43.17 cells were double positive for YFP+/CgA+ in comparison with stressed mice where 113.5 cells were double positive for YFP+/CgA+, total number of cells). Populations of YFP+/S100+ (glia) and neuronal (YFP+/β‐III tubulin+) cells was similar in both experimental groups (Supporting Information Fig. S6). (C‐D): Numbers are expressed as mean ± SD. Statistical significance was determined using Student's t test.; *, p < 0.05. (E): In summary, under basal conditions the glia‐like, nestin–GFP expressing stem cells express the progenitor markers nestin and Prominin‐1, as well as the glia markers glial fibrillary acidic protein and S100b. These cells are almost quiescent under basal conditions. Under stress conditions, immobilization stress in this case, a fraction of the nestin–GFP expressing glia‐like progenitors proliferate and differentiate towards the specific cells required to cope with stress, predominantly into the chromaffin lineage; also differentiation into neuron‐like cells was observed. Abbreviations: CgA, chromogranin A; GFAP, glial fibrillary acidic protein. To characterize the differentiated progeny of nestin–expressing progenitor cells in long‐term stress, tamoxifen‐induced nestin–CreER:Rosa26‐YFP mice were subjected to the identical immobilization protocol applied to the nestin–GFP mouse (Fig. 7B). Immobilization stress resulted in the same decrease in body weight (Supporting Information Fig. S5A) and increase in adrenal gland weight (Supporting Information Fig. S5B; n = 6) as observed in the nestin–GFP model. The number of YFP‐positive cells was significantly increased in the stressed mice, suggesting that nestin–expressing progenitors were activated and differentiated to cope with the physiological situation (Fig. 7C). Regarding the glia lineage (coexpression of GFAP and S100b) as well as neuronal lineage (β‐III tubulin+/YFP+) no significant differences were found between control and stress mice (Fig. 7D). However, the number of YFP positive cells coexpressing CgA was significantly increased in the stressed group (Fig. 7D). As an absolute control for these induction experiments, the adrenal medullae of nestin–CreER:Rosa26‐YFP mice were immunohistologically analyzed immediately after treating the animals with tamoxifen under basal conditions for 2 consecutive days. In accordance with the findings from the nestin–GFP mouse model, under these conditions, the majority of YFP‐expressing cells belonged to the glia lineage (83.12 ± 2.51% of total YFP+ cells were positive for S100b). A small percentage of YFP+ cells related to the chromaffin lineage (16.88 ± 2.51% of total YFP+ cells were positive for CgA) (Supporting Information Fig. S7). The existence of YFP‐expressing chromaffin cells under these basal conditions might be a consequence of the stress implicated by the tamoxifen injections or may represent the basal differentiation that occurs even in unstressed animals. These cell‐lineage tracing stress experiments demonstrate that under chronic stress conditions nestin‐expressing progenitor cells in the adrenal medulla differentiate to adapt the gland to the increased physiological needs, predominantly into chromaffin cells (Fig. 7D). Discussion Nestin–GFP Progenitor Cells in the Adult Adrenal Medulla In this study, we show that a defined pool of nestin–GFP expressing progenitor cells exists in the adult adrenal medulla. These cells have glial properties and features of sustentacular cells, the glia population in adrenal medulla. These glia‐like cells are multipotent and, in addition to the glial lineage, differentiate into the two major lineages relevant to adrenal function, chromaffin cells and neurons. Furthermore, our results show their involvement in the gland's response and adaptation to long‐term stress. Due to the close relation and similarities of the adrenal medulla with neural tissue on the one hand—chromaffin cells are modified post‐ganglionic sympathetic neurons—and its high plasticity on the other hand, the adrenal medulla has been extensively studied [44]. Although the high plasticity suggests the involvement of progenitor cells, their involvement especially in stress adaptation has not been studied so far. Our previous data suggest the presence of progenitor cells in adult bovine and human adrenal medulla [9, 10]; however, these two models did not allow addressing the cells' localization within the gland, studying their role in vivo and tracing their progeny. Our present results establish the persistence of nestin–GFP expressing cells in the adult adrenal representing a reservoir of undifferentiated progenitors/stem cells. These cells are intermingled with mature chromaffin cells but clearly represent a distinct population since they were characterized by different morphology and did not coexpress chromaffin cell specific markers, such as CgA or TH. We explored their proliferative potential and conclude that in vivo these progenitor cells show a very low proliferative activity under basal physiological conditions with EdU incorporation in only approximately 1% of nestin–GFP positive cells. On the other hand, nestin–GFP positive cells are able to proliferate in vitro, where they are not under influence of their “stem cell niche.” This is similar to neural stem cells [45, 46]. In this respect, adrenomedullary nestin–GFP progenitors seem to share features with progenitors in adult brain. Our results show coexpression of nestin–GFP and S100b; furthermore, 62% of the cells expressed the radial glia marker GFAP. The three major adrenomedullary cell types are chromaffin cells, postganglionic neurons, and supportive sustentacular cells [47]. Sustentacular cells share properties with Schwann and satellite cells [34] and are characterized by S100b expression [34-36]. This study shows that all S100b‐positive cells also expressed nestin–GFP, indicating their progenitor properties. Thus, adrenomedullary sustentacular cells constitute a population with stem cell properties contributing to the tissue's high plasticity. In this respect, nestin–GFP cells in adult adrenal medulla share similarities with radial glia progenitors in adult brain by coexpressing glial markers and being quiescent under normal conditions [21, 48]. Similar to the adrenal medulla another neural‐like tissue, the olfactory epithelium, has the ability to regenerate throughout life. Also here, glia‐like sustentacular cells express nestin, have stem cell properties, and resemble CNS neuroglia in rat [49] and human [50]. Neurons comprise another major cellular part of the adrenal medulla. Chromaffin cells are excitable neuron‐like cells with numerous afferent and efferent connections. Furthermore, the adrenal medulla is characterized by intraadrenal ganglionic neurons that innervate chromaffin cells and the adrenal cortex [47, 51]. Our results also indicate the neural differentiation of nestin–GFP cells by coexpressing β‐III tubulin in some cells. However, progenitors coexpressing this neuronal marker were characterized by a GFP‐signal of low intensity indicating downregulation of nestin and consequently GFP due to differentiation. This is in accordance with the developing brain of the same animal model where decreased nestin–GFP transgene expression was observed in the progeny of neuroepithelial cells entering the path of neuronal differentiation resulting in Tuj1 expression in GFP‐dim cells only while GFAP‐expression was localized in GFP‐bright cells [17]. The adrenal is highly vascularized to carry secretory products into systemic circulation and to receive information about the organism's physiological status. We found nestin‐expressing progenitors with processes reaching blood vessels thus allowing close interactions with endothelial cells and blood‐derived factors. These interactions are probably important in controlling these progenitors' activation. Interestingly, this close contact to blood vessels also seems a morphological feature shared with NSC [46]. Nestin+ Progenitor Cells Are Differentiation‐ and Proliferation‐Competent Nestin–GFP positive progenitor cells in vitro formed spheres—chromospheres—and proliferated similar to brain NSC in neurospheres [52]. In contrast to their low proliferation rate in vivo, adrenomedullary nestin‐progenitors proliferated in vitro as indicated by EdU incorporation within chromospheres, where 66% of EdU labeled cells were nestin–GFP positive. Thus, with prolonged time in culture chromospheres were enriched in nestin–GFP progenitors resulting in a homogeneous population. In vitro differentiating progenitor cells coexpressed the glia markers GFAP and S100b. In accordance with our in situ observations, the neuronal marker β‐III tubulin labeled GFP‐dim cells with neuron‐like morphology, indicating their neuronal differentiation. Proof for the differentiation potential to all three lineages compiling the adrenal medulla, however, came from the nestin–CreER:Rosa26‐YFP mice. In vitro induction of YFP‐expression by Cre‐recombination in adrenomedullary progenitors demonstrated their differentiation to the three cell populations required in adrenal medulla, chromaffin, neuronal, and sustentacular cells: they coexpressed CgA, which is the major protein in chromaffin vesicles and is thus specific for differentiated chromaffin cells; the intermediate filament β‐III tubulin specific for early and differentiated neurons; or S100b, respectively. In vivo nestin–CreER lineage tracing confirmed the differentiation into these three lineages. These results verify the generation of newly differentiated cells in the adult adrenal medulla and indicate their derivation from nestin‐positive multipotent stem cells. Together with the observations from nestin–GFP mice these data show the existence of a population of nestin‐expressing progenitors with glial properties that activate when required. The close similarity of chromaffin cells to neurons has promoted their potential use in transplantation therapies of neurodegenerative diseases (reviewed in ref. [53, 54]). The proliferation and differentiation of adrenomedullary progenitor cells into functional neurons might be a new promising strategy in regenerative treatments of neurodegenerative diseases. Our results indicating that pro‐neural culture conditions induce a pure neuronal and glia differentiation of progenitor cells from secondary spheres, together with our previous observation that nestin positive progenitors exist in the human adult adrenal medulla [10], further supports the clinical potential of these cells. The glia‐like properties of the nestin–GFP positive stem cells and their neural differentiation potential might also explain the frequent observation of pheochromocytomas with sustentacular properties [55, 56]. This dual differentiation was also observed in the rat pheochromocytoma cell line PC12 although it is supposed to be monoclonal [57], suggesting that PC12 cells might be derived from a multipotent adrenomedullary precursor cell with properties characterized here. Immobilization Stress Promotes Differentiation of Progenitor Cells The adrenal medulla has a central role in the body's reaction to stress and the proper adaptation of adrenomedullary function is central in the body's stress response and regain of homeostasis [5]. Our results show that progenitor cells in the adrenal medulla are activated by the highly demanding physiological situation of repeated immobilization and contribute to the gland's adaptation. On day 1 of the immobilization stress, the number of EdU+/ nestin–GFP+ cells together with S100b+/ nestin–GFP+ cells had already increased, suggesting that under acute stress conditions the adrenal medulla copes by increasing the population of glia cells supporting chromaffin cells. This is in accordance with the adrenal medulla's physiological function in the short term response to stressful situations. However, the number of nestin–GFP progenitor cells significantly decreased during the six days of immobilization stress. The results, with a decline of GFAP+/ nestin–GFP+ cells and a significant increase of S100b+/ nestin–GFP+ cells after one day of immobilization stress suggest that nestin–GFP progenitors in the adrenal medulla behave as activated mature glia cells during shorter periods of stress. These sustentacular cells might be important in maintaining the highly activated chromaffin cells during these short periods. But when the stress becomes chronic, this supportive glial differentiation might not be sufficient to cope with the requirements leading to a reduction of S100b+/ nestin–GFP+ cells resulting in a significant decrease of the GFP+ population at the end of the stress period. These data, together with the finding that EdU+/ nestin–GFP+ cells were increased at the first day of stress suggest that nestin‐progenitors proliferated and differentiated, thereby reducing nestin‐and thus GFP‐expression. The pool of nestin–GFP+ cells had recovered after seven days of resting. Lineage tracing in nestin–CreER:Rosa26‐YFP mice confirmed the differentiation. Under normal conditions, progenitor cells mainly differentiated into glia cells; a small percentage differentiated into Tuj1 expressing neurons. Conversely, under stress conditions differentiation moved significantly toward the chromaffin lineage resulting in CgA‐expressing chromaffin cells; the number of glial cells compared with control conditions was unchanged. Interestingly, newly formed glial cells characterized by S100b expression were never found in proximity to the newly form chromaffin cell clusters suggesting a supportive function for “old” chromaffin cells. The effect of immobilization stress on adrenomedullary function has been extensively studied. It has been shown that immobilization stress for up to 2 hours is accompanied by a decrease in adrenomedullary epinephrine content while repeated immobilization resulted in normal or even higher basal catecholamine levels [8]. This was attributed to an increased capacity to synthesize catecholamines mainly due to increased expression of phenylethanolamine N‐methyltransferase, the enzyme converting norepinephrine to epinephrine [8, 58]. However, in addition to this molecular adaptation our results confirm an adaptation on the cellular level by recruiting new chromaffin cells from the stem cell pool within the adult adrenal medulla to cope with this highly demanding situation. Conclusion In conclusion, our data localize glia‐like nestin expressing progenitor cells in the adult adrenal medulla that might correspond to sustentacular cells. These progenitors, although mainly quiescent in vivo, are proliferation and differentiation competent and are able to give rise to chromaffin cells and neurons. Stress experiments allowed us to understand the involvement of these progenitors in the adrenal's response to stress by forming new chromaffin cells (Fig. 7E). This probably is central to the gland's high plasticity necessary to adapt to different physiological and pathological situations. In addition to being the first report demonstrating the involvement of a multipotent stem cell population of glia‐like cells in adrenal medullary tissue formation and its adaptation to physiological needs, our study sheds light on the contribution of stem and progenitor cells in the adaptation of neuroendocrine tissue function and might be extended to other neuroendocrine tissues. Acknowledgments We thank Manuela Rothe and the animal facility at the MTZ Dresden for taking care of the experimental animals and Kathleen Eisenhofer for language editing. This work was supported by funds from the Deutsche Forschungsgemeinschaft Clinical Research Unit KFO 252 “Microenvironment of the Adrenal in Health and Disease” (to M.E.‐B., T.C., A.A.‐T., and S.R.B.) and SFB 655 “From cells to tissues” (to M.E.‐B. and S.R.B.) and the Center for Regenerative Therapies, Dresden, Germany. 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Google Scholar Crossref Search ADS PubMed WorldCat © 2015 AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Multipotent Glia‐Like Stem Cells Mediate Stress Adaptation
JO - Stem Cells
DO - 10.1002/stem.2002
DA - 2015-06-01
UR - https://www.deepdyve.com/lp/oxford-university-press/multipotent-glia-like-stem-cells-mediate-stress-adaptation-fmmKlZLM5O
SP - 2037
EP - 2051
VL - 33
IS - 6
DP - DeepDyve
ER -