Adrenomedullary progenitor cells: Isolation and characterization of a multi-potent progenitor cell population

Molecular and Cellular Endocrinology 408 (2015) 178–184

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Molecular and Cellular Endocrinology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m c e

Review

Adrenomedullary progenitor cells: Isolation and characterization of a multi-potent progenitor cell population Vladimir Vukicevic a, Maria Fernandez Rubin de Celis a, Natalia S. Pellegata b, Stefan R. Bornstein c,d, Andreas Androutsellis-Theotokis d,e, Monika Ehrhart-Bornstein a,d,* a

Division of Molecular Endocrinology, Medical Clinic III, Carl Gustav Carus University Clinic, Technische Universität Dresden, 01307 Dresden, Germany Institute of Pathology, Helmholtz Zentrum München, 85764 Neuherberg, Germany c Medical Clinic III, Carl Gustav Carus University Clinic, Technische Universität Dresden, 01307 Dresden, Germany d Center for Regenerative Therapies Dresden, Technische Universität Dresden, 01307 Dresden, Germany e Division of Stem Cell Biology, Medical Clinic III, Carl Gustav Carus University Clinic, Technische Universität Dresden, 01307 Dresden, Germany b

A R T I C L E

I N F O

Article history: Received 31 October 2014 Received in revised form 25 December 2014 Accepted 27 December 2014 Available online 6 January 2015 Keywords: Adrenal medulla Sympathoadrenal Stem cells Progenitor cells ALDH Hes3

A B S T R A C T

The adrenal is a highly plastic organ with the ability to adjust to physiological needs by adapting hormone production but also by generating and regenerating both adrenocortical and adrenomedullary tissue. It is now apparent that many adult tissues maintain stem and progenitor cells that contribute to their maintenance and adaptation. Research from the last years has proven the existence of stem and progenitor cells also in the adult adrenal medulla throughout life. These cells maintain some neural crest properties and have the potential to differentiate to the endocrine and neural lineages. In this article, we discuss the evidence for the existence of adrenomedullary multi potent progenitor cells, their isolation and characterization, their differentiation potential as well as their clinical potential in transplantation therapies but also in pathophysiology. © 2014 Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2.

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Introduction ........................................................................................................................................................................................................................................................ Sympathoadrenal progenitor cells in the adrenal medulla ................................................................................................................................................................ 2.1. Are sympathoadrenal progenitor cells maintained in the adult adrenal medulla? ..................................................................................................... 2.2. Isolation and characterization of sympathoadrenal progenitors ........................................................................................................................................ 2.3. Intra-adrenal regulation of sympathoadrenal progenitor differentiation ........................................................................................................................ Model for in vitro studies ............................................................................................................................................................................................................................... Potential involvement of sympathoadrenal progenitor cells in tumorigenesis .......................................................................................................................... Potential cell source for transplantation therapies ............................................................................................................................................................................... Conclusions ......................................................................................................................................................................................................................................................... Acknowledgements .......................................................................................................................................................................................................................................... References ............................................................................................................................................................................................................................................................

1. Introduction The adrenal gland is composed of two endocrine tissues of different embryologic origin, the mesodermally derived adrenal cortex

* Corresponding author. Molecular Endocrinology, Medical Clinic III, Carl Gustav Carus University Clinic, Technische Universität Dresden, Fetscherstr. 74, 01307 Dresden, Germany. Tel.: +49 351 4586130; fax: +49 351 4587334. E-mail address: [email protected] (M. EhrhartBornstein). http://dx.doi.org/10.1016/j.mce.2014.12.020 0303-7207/© 2014 Elsevier Ireland Ltd. All rights reserved.

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and the neural crest derived chromaffin cells of the adrenal medulla. The adrenal is thus particularly suitable to study tissue development, the tissue microenvironment and interactions of different cell types during development and adaptation to physiological needs. We previously demonstrated the critical role of cellular interactions of both steroid and catecholamine producing cells in adrenal physiology and disease (Bornstein et al., 2008; Ehrhart-Bornstein et al., 1998). Furthermore, both adrenal cortex and medulla are highly plastic and able to adapt to physiological needs, suggesting the involvement of progenitor cells. Intensive work within the last years

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has identified the involvement of adrenocortical stem cells in the regeneration and adaptation of the adult adrenal cortex (references in this issue; Walczak and Hammer, 2015). Little, however, is known on progenitor cells in the adrenal medulla, their proliferation and differentiation.

fact that Sox10 expression is down-regulated in the adult adrenal medulla, its importance was thought to be restricted to adrenal medulla development (Reiprich et al., 2008). Our data, specifically the expression of SoxE genes in the adult adrenal medulla, however, suggest the persistence of neural crest derived progenitor cells.

2. Sympathoadrenal progenitor cells in the adrenal medulla

2.2. Isolation and characterization of sympathoadrenal progenitors

2.1. Are sympathoadrenal progenitor cells maintained in the adult adrenal medulla?

The isolation of sympathoadrenal progenitor cells is one important goal in order to study their properties in a homogenous culture. Several flow cytometry methods were used to identify and isolate stem cells based on metabolic activity. The expression of high levels of aldehyde dehydrogenase (ALDH) activity and staining with a fluorescent substrate for ALDH has first been described to identify and isolate primitive hematopoietic stem cells (Fallon et al., 2003; Jones et al., 1995) and ALDH-bright (ALDHbr) cell populations with stem cell activity have in recent years been sorted from several normal tissues (Balber, 2011). The ALDH expression assay has also been adapted to effectively identify and isolate neural stem cells (NSCs) and progenitors from adult and embryonic murine neurospheres and dissociated tissue (Corti et al., 2006; Obermair et al., 2010). Based on these previous reports, we assumed that medullary sympathoadrenal progenitor cells might contain high levels of ALDH that could be used as a novel identification marker for their isolation and characterization. The fraction of sympathoadrenal progenitor cells was enriched from chromospheres based on the intracellular ALDH activity using the ALDEFLUOR (StemCell Technologies, Köln, Germany) method (Balber, 2011). The progenitor population was detected and isolated by flow cytometry (FACSAria, Becton-Dickinson, Franklin Lakes, NJ, USA) due to their pronounced activity of ALDH resulting in higher intensity of fluorescence (ALDHbr) and characteristic low granularity displayed as Side Scatter Low (ALDHbrSSClo) (Fig. 1). To investigate the gene expression profile in this potential progenitor cell population, a microarray analysis of gene expression was performed in isolated progenitors (ALDHbrSSClo) sorted compared with ALDHlow chromosphere cells (Fig. 2). On the one hand, several genes characteristic of neural or neural crest progenitor and stem cells were detected in the ALDHbrSSClo population. Up-regulation of Notch2 and its downstream effector Hes1 has been described in neural stem cells (Borghese et al., 2010; Solecki et al., 2001), preventing differentiation and promoting self-renewal. The expression in the ALDH br SSC lo population suggests a similar function in sympathoadrenal progenitor cells. This is in accordance with our previous data showing Notch2 and Hes1 expression in chromosphere cells (Vukicevic et al., 2012b). The expression of genes involved in other pathways indicates diversity of molecular mechanisms in the regulation of sympathoadrenal progenitor cells. This for example includes Wnt2B ligand that, as a part of the canonical pathway, plays an important role in the regulation of neural crest progenitor cell differentiation during eye development (Grocott et al., 2011; Kubo et al., 2005). Upregulation of WLS and WISP2 genes is considered to contribute to the control of stem cell renewal downstream of Wnt (Wend et al., 2010). Moreover, isolated sympathoadrenal progenitors displayed upregulated homebox transcriptional repressor MSX1 which is involved in the regulation of neural crest specification and is particularly expressed in neural ectoderm (Gammill and Bronner-Fraser, 2003; Ishii et al., 2005). On the other hand, genes characteristic for differentiating or differentiated neural and/or chromaffin cells were downregulated. These include alpha and beta tubulins involved in neural differentiation. Moreover, genes involved in the differentiation of sympathoadrenal progenitors such as ASCL1 (MASH1) (Huber et al., 2002), GATA2 (Tsarovina et al., 2004) and HAND1 (Vincentz et al., 2012) and genes involved in catecholamine synthesis such as

Chromaffin cells of the adrenal medulla develop from neural crest stem cells. Stem cells are defined as an entity with unlimited selfrenewal and the capacity to give rise to multiple cell types (Seaberg and van der Kooy, 2003; Weiner, 2008) while progenitor cells possess a limited self-renewal capacity and are often uni- or multipotent. Together with sympathetic neurons of the dorsal ganglia and the intermediate small intensely fluorescent (SIF) cells chromaffin cells develop from a common sympathoadrenal progenitor (Shtukmaster et al., 2013). Along their migratory route, sympathoadrenal progenitors become catecholaminergic, aggregate at the dorsal aorta, and then further migrate to the adrenal anlagen, where they differentiate into mature chromaffin cells (reviewed in Huber et al., 2009). It is now apparent that various adult tissues maintain neural crestderived multipotent progenitor cells (for example: Brandl et al., 2009; Davies et al., 2010; Sieber-Blum and Hu, 2008; Widera et al., 2009). Multipotent progenitor cells are often considered adult stem cells. This, however, neglects the functional properties of progenitor cells gained during differentiation that render them distinct from stem cells. Already 30 years ago, SIF cells, the third cell type in the sympathoadrenal branch of the neural crest lineage which exist primarily as a minority cell population in autonomic ganglia, were suggested to constitute sympathoadrenal progenitors that persist in the adult and retain their stem cell properties. These cells exhibit a progenitor phenotype and progenitor properties reflected by their potential to differentiate to sympathetic neurons and chromaffin cells (Doupe et al., 1985a, 1985b). If neural crest-derived sympathoadrenal progenitors are maintained in adult tissues such as ganglia, these progenitor cells might also persist in the adult adrenal medulla where they could contribute to the gland’s adaptation to physiological needs. Work in recent years from our group indeed proved the existence of sympathoadrenal progenitor cells in the adult adrenal medulla. Sympathoadrenal and neural progenitors share some properties in terms of gene expression patterns, lower self-renewal throughout life and their ability to differentiate into functional neurons in vitro. Based on this, cells with progenitor properties were first identified in and enriched from bovine adrenal medulla (Chung et al., 2009). Similar to neural stem cells, these progenitor cells, when prevented from adherence to the culture dish, grow in suspension as free-floating spheres with self-renewing capacity, which we named chromospheres. Importantly, sympathoadrenal progenitor cells could also be identified in the adult human adrenal medulla and cultured in vitro (Santana et al., 2012). The progenitor cells from adrenal medulla which were enriched in chromospheres share significant properties with neural stem cells, expressing specific stem cell markers including neural stem cell markers such as nestin, CD133, Notch1, nerve growth factor receptor, musashi1, Snai2, Sox9 and Sox10 (Chung et al., 2009; Santana et al., 2012; Vukicevic et al., 2012b) and proteins of the Notch pathway (Vukicevic et al., 2012a). Sox9 and Sox10 are members of the SoxE subgroup, which play an important role in the migration and differentiation of neural crest derivatives (Cheung and Briscoe, 2003; Cheung et al., 2005; Kim et al., 2003) and the specification and survival of chromaffin precursor cells during embryonic development (Reiprich et al., 2008). Due to the

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ALDH Fig. 1. Flow cytometry for aldehyde dehydrogenase (ALDH) activity in adrenomedullary chromosphere cells; preceding analysis, cells were cultured in sphere culture for 7 days. The progenitor-stem cell population is presumed to display high levels of aldehyde dehydrogenase – ALDH and low inner granularity SSClow. (A) Unstained sample; (B) negative control, obtained by staining with ALDEFLUOR and addition of a specific inhibitor of ALDH activity, diethylaminobenzaldehyde (DEAB) to inhibit substrate cleavage; (C) a heterogeneous population, probably consisting of progenitor and stem cells was identified upon addition of ALDEFLUOR substrate for ALDH (ALDHbrSSClo cells within region, white arrow). Measurement was performed on a BD FACScalibur flow cytometer equipped with a 488 nm argon-ion laser for excitation. Emission was detected at 515–545 nm filter.

phenylethanolamine-N-methyltransferase (PNMT), tyrosine hydroxylase (TH) and dopamine-β-hydroxylase (DBH) were significantly downregulated. These data strongly indicate the existence of sympathoadrenal progenitor cells with neural stem cell characteristics which can be isolated by FACS due to their ALDH expression. 2.3. Intra-adrenal regulation of sympathoadrenal progenitor differentiation How is the differentiation of adrenomedullary SA progenitor cells regulated? During development, the differentiation of neural crest derived stem cells into chromaffin cells and sympathetic neurons is regulated by a network of local factors (Huber et al., 2009; Morigushi et al., 2007). In the center of the signaling cascade that coordinates neural crest cell migration and cell lineage segregation are morphogenic factors synthesized by the dorsal aorta. These include bone morphogenetic proteins (BMPs), including BMP4, which play a central role in the induction of a catecholaminergic fate (Saito et al., 2012). From the moment sympathoadrenal progenitors invade the adrenal anlagen their differentiation is under the influence of factors released from the fetal and later the adult adrenal cortex. Which are the adrenocortical factors that might regulate sympathoadrenal progenitor cells? In the human embryo, sympathoadrenal progenitor cells surrounding and invading the human adrenal anlagen are already present at the gestational age of 6 weeks (Ehrhart-Bornstein et al., 1997; Jozan et al., 2007). An influence of this adrenal primordium, the fetal adrenal cortex, on the endocrine differentiation of the invading neural crest-derived sympathoadrenal progenitor cells has long been discussed. Based on in vitro data showing that glucocorticoids promote the endocrine differentiation of chromaffin cells and the fact that the expression of phenylethanolamineN-methyltransferase (PNMT), the enzyme converting norepinephrine to epinephrine, depends on high glucocorticoid concentrations (Wurtman and Axelrod, 1966), a central role in the differentiation of chromaffin cells had been attributed to adrenocortical glucocorticoids. In contrast to these in vitro observations, data from

glucocorticoid receptor knockout mice (Finotto et al., 1999) and from steroidogenic factor-1 (SF-1) knockout mice (Luo et al., 1994) revealed the development of chromaffin cells independent of glucocorticoids (Finotto et al., 1999; Gut et al., 2005). These chromaffin cells, however, did not express PNMT and consequently were unable to produce epinephrine. Thus, chromaffin cells develop their endocrine phenotype even in the absence of glucocorticoids suggesting the involvement of other, probably intra-adrenal factors. In this context it is crucial to note that sympathoadrenal progenitor cells persist in the adult adrenal medulla despite the high local concentrations of glucocorticoids furthermore challenging the central role of glucocorticoids in their differentiation. Of course it is conceivable that the local microenvironment of progenitors protects them from the action of glucocorticoids. However, in in vitro cell culture conditions, sympathoadrenal progenitors were present in chromospheres despite the presence of glucocorticoids in the culture medium (Vukicevic et al., 2012b), indicating in accordance with data from glucocorticoid receptor knockout mice (Finotto et al., 1999) that factors other than glucocorticoids are necessary to induce the endocrine differentiation of these progenitor cells. The fetal adrenal cortex does not produce relevant levels of glucocorticoids; the major steroid produced instead is dehydroepiandrosterone sulfate (DHEAS); DHEAS is converted to estradiol in the placenta for sustaining pregnancy (Keegan and Hammer, 2002; Mesiano and Jaffe, 1997). DHEA influences adult chromaffin progenitor cells by reducing their proliferation and stimulating their differentiation (Chung et al., 2011) suggesting the involvement of adrenal androgens in the differentiation process of sympathoadrenal progenitor cells also during development. In the adult bovine and human adrenal DHEA is mainly synthesized and secreted from the zona reticularis. The close contact between the zona reticularis and the adrenal medulla suggests high local concentrations of DHEA in the adrenal medulla and physiological interactions between the two endocrine tissues (Bornstein et al., 2008; Ehrhart-Bornstein et al., 1998). DHEA and DHEAS in

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Fig. 2. Cluster analysis of gene expression in chromaffin progenitor cells from ALDHbr(left column) versus ALDHlow (right column). Genes found to be reproducibly upregulated in ALDHbr population (progenitor cells with higher brightness) compared with ALDHlow population (chromosphere cells with lower ALDH brightness) are highlighted in green; those expressed at a lower level are in red. Differences in gene expression between ALDHbr vs ALDHlow are presented as log2 of fold change (n = 3). RNA extraction and microarray hybridization: Total RNA was isolated using RNeasy Plus Mini kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. Quality control was performed by using the Agilent Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA, USA). The hybridization cocktail containing cDNAs labeled with biotin was added to the Affymetrix GeneChip Bovine Genome Array. Hybridizations on the Affymetrix platform were performed in triplicate with labeled cDNA comparing ALDHbr vs ALDHlow samples. Microarray data analysis: The custom analysis of the microarray data for selected genes was processed through Linear Models for Microarray Data (Limma) and Affy package (Bioconductor, Open software tools for bioinformatics, http://www.bioconductor.org). Robust Multichip Analysis normalization was utilized by Affy package’s GCRMA (GeneChip RMA) method. The fold change between ALDHbr and ALDHlow was estimated by fitting a linear model for each gene and “Bayes” moderation of the standard error is performed with eBayes. Values higher than 1.5 fold were selected for further analysis. The statistical analysis was performed by Benjamini and Hochberg testing method which controls the false discover rate to minimize the percentage of false positives. Determined difference in gene expression (ALDHbr vs ALDHlow) was considered significant when p ≤ 0.05, n = 3 for each group.

addition differentially regulate growth-factor induced proliferation of adult bovine chromaffin cells (Sicard et al., 2007) and of the proliferation and differentiation of the rat pheochromocytoma cell line PC12 (Krug et al., 2009; Ziegler et al., 2008, 2011) further indicating an influence of these adrenocortical steroids on the adrenal medulla. This is further supported by the observation that in congenital adrenal hyperplasia in patients with classic 21-hydroxylase deficiency chromaffin cell development is altered. An influence on the adrenal medulla of the high concentrations of androgens in addition to the low levels of glucocorticoids in these patients had also been proposed (Merke and Bornstein, 2005; Merke et al., 2000). BMP4, another factor centrally involved in regulating sympathoadrenal differentiation during embryologic development, is, in addition to the dorsal aorta, produced by the adrenal cortex (Huber et al., 2008) and thus might constitute another intraadrenal factor influencing in a paracrine manner the differentiation

of sympathoadrenal stem cells during development but also in the adult adrenal. This is supported by our observation that BMP4 influences the proliferation and differentiation of adult sympathoadrenal progenitor cells (Chung et al., 2009, 2011). These in vitro data suggest that BMP4 stimulates the proliferation of these cells while restricting their differentiation and reducing the expression of catecholaminergic enzymes. In accordance, BMP4 did not stimulate tyrosine hydroxylase expression and dopaminergic differentiation of Neuro 2A cells, a mouse neural crest-derived cell line (Tremblay et al., 2010). In summary, the differentiation of sympathoadrenal progenitors located within the adrenal during development but also in the adult seems, at least in part, to be regulated by local factors released from the adrenal cortex in addition to glucocorticoids which are indispensable for the induction of the adrenergic phenotype but do not seem to be relevant for the induction of differentiation.

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3. Model for in vitro studies Chromospheres are heterogeneous structures (Chung et al., 2009). Homogenous cultures, however, are advantageous to study and manipulate the properties of these cells. Cell culture media with defined compositions are also advantageous as they omit unknown factors that may confound interpretation of data. Therefore, monolayer, serum-free culture techniques developed for central nervous system neural stem cells were adapted to the culture of sympathoadrenal progenitor cells of the adrenal medulla (Masjkur et al., 2014). Such cell culture conditions are valuable for neural stem cell research because they can allow the modeling of particular signal transduction pathways that are also operational in vivo. Specifically, they maintain the operation of the STAT3-Ser/Hes3 Signaling Axis, a newly elucidated signal transduction pathway regulating the number of central nervous system stem cells in vitro and in vivo (Androutsellis-Theotokis et al., 2006, 2010). At the heart of this signaling pathway are two key events: The phosphorylation of STAT3 (Signal transducer and activator of transcription 3) and the subsequent transcription of the transcription factor Hes3 (Hairy and enhancer of split 3). A number of pharmacological treatments promote these events, including basic Fibroblast Growth Factor (bFGF), insulin, soluble forms of Notch receptor ligands, the Tie2 receptor ligand Angiopoietin 2, and cholera toxin. The pathway is opposed by Janus Kinase (JAK) activity; therefore, inclusion of a JAK inhibitor promotes the pathway and cell yield. These treatments increase endogenous neural stem cell number in vitro and in vivo. We recently demonstrated that Hes3 is expressed in the adult bovine adrenal medulla as well as in putative chromaffin progenitor cultures, using the new monolayer culture system devoid of serum (and hence, unknown factors). These data suggest the operation of this signaling pathway also in adult adrenomedullary progenitors. To show the functional relevance of these observations, we treated these cells with a variety of activators of the STA3-Ser/Hes3 Signaling Axis, including a soluble form of the Notch receptor ligand Delta4, Angiopoietin 2, a JAK inhibitor, a combination of the three factors, and cholera toxin. All these treatments showed a tendency toward increased cell yield, with the combination treatment reaching significant values, providing evidence for the functional operation of this pathway in adrenomedullary chromaffin progenitors, introducing new putative regulators of adrenomedullary function (Masjkur et al., 2014). These data further support the similarity of adrenomedullary sympathoadrenal progenitor cells and neural stem cells from the central nervous system. 4. Potential involvement of sympathoadrenal progenitor cells in tumorigenesis The identity of the cell of origin of a tumor is still unsolved (Magee et al., 2012). It has been suggested that immature cells, including neural stem cells, require fewer genetic alterations than mature cells in order to turn into tumor initiating cells (Huse and Holland, 2010). It is thus conceivable that in a tissue with high numbers of progenitor cells, such as the adrenal medulla, progenitor cells might be involved in the process of tumorigenesis. Pheochromocytomas are catecholamine-producing neuroendocrine tumors arising from chromaffin tissue of the adrenal medulla (Lenders et al., 2005). They are highly variable depending in part on the underlying mutations in hereditary forms of the tumor and the associated differences in catecholamine biosynthetic and secretory pathways (Eisenhofer et al., 2001, 2004; Qin et al., 2014). Increasing evidence indicates that these tumors, at least in part, may derive from persistent sympathoadrenal progenitor cells within the adrenal medulla. This hypothesis is based on the fact that pheochromocytomas overexpress multiple genes that are involved in early neural development (Molatore et al., 2010; Powers et al., 2007; Qin

et al., 2014) and that the expression of some of these genes also characterize sympathoadrenal progenitor cells from the adult adrenal medulla (Chung et al., 2009; Ehrhart-Bornstein et al., 2010; Santana et al., 2012). In addition to forming the adrenal medulla, neural crest derived sympathoadrenal progenitors also play an important role in the development of the sympathetic nervous system. An important pathology of the sympathetic peripheral nervous system is neuroblastoma (Weiss et al., 1997). Data from transgenic mouse models suggest sympathoadrenal progenitors as putative origin of this pathology. Based on our data, cultures of sympathoadrenal progenitor cells from postnatal rodent adrenals were recently established to provide an in vitro model to study the pathological and aberrant development of sympathoadrenal progenitor cells leading to the development of neuroblastoma (Saxena et al., 2013). 5. Potential cell source for transplantation therapies Mounting evidence suggests multipotent properties of the neural crest-derived sympathoadrenal progenitors; these cells are able to differentiate in vitro to cells of the endocrine and the neural lineage, specifically to mature catecholaminergic neurons (Chung et al., 2009; Santana et al., 2012; Vukicevic et al., 2012b, 2015). These data indeed suggest the sympathoadrenal progenitors’ involvement in the adaptation and regeneration of adrenomedullary tissue. In addition, these cells might bear potential in the regenerative treatment of neurodegenerative diseases. Due to the close relation of chromaffin cells to catecholaminergic neurons, a substantial number of studies have promoted the use of chromaffin cells in the treatment of neurodegenerative disorders, such as Parkinson’s disease (reviewed in Ambriz-Tututi et al., 2012). Between 1988 and 2001, more than 300 Parkinsonian patients were treated by autologous adrenal transplants with some improvement of the clinical symptoms. Survival rates of grafted adult chromaffin cells, however, were limited and clinical improvements disappeared 1–2 years after transplantation (Fernandez-Espejo et al., 2005). A serious disadvantage in the usage of adult adrenal medulla possibly was the postmitotic nature of most transplanted cells. The identification and isolation of sympathoadrenal progenitor cells from the human adrenal medulla (Santana et al., 2012) might ignite new hopes for their potential use in the treatment of neurodegenerative diseases such as Parkinson’s disease. 6. Conclusions In summary, sympathoadrenal progenitor and stem cells exist in the adrenal medulla. Their differentiation into chromaffin cells seems to be under the local, intra-adrenal influence of factors including growth factors and hormones released from the adrenal cortex. The isolation and characterization of these cells provides the basis to study their involvement in the gland’s adaptation to physiological needs. Furthermore, specific culture conditions allow their manipulation and the study of potential pathologic developments in vitro. Finally, the isolation of these neural stem cell-like progenitor cells is the prerequisite for their use in transplantation therapies of neurodegenerative diseases. Acknowledgements This work was supported by funds from the Deutsche Forschungsgemeinschaft: Clinical Research Unit KFO 252 “Microenvironment of the Adrenal in Health and Disease” to MEB, AAT and SRB, SFB 655 “From cells to tissues” to MEB and SRB, DFG-SFB824, subproject B08 to NSP and the Center for Regenerative Therapies, Dresden, Germany. The authors declare that no conflict of interest exists.

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