Vladimir Vukicevic

Age-dependent neuroectodermal differentiation capacity of human mesenchymal stromal cells: limitations for autologous cell replacement strategies

BACKGROUND AIMS Human adult bone marrow (BM)-derived mesenchymal stromal cells (hMSC) are reported to break germ layer commitment and differentiate into cells expressing neuroectodermal properties. Although it is of pivotal interest for cell replacement therapies for neurologic disorders, no data exist on the influence of the donors age on this multipotent differentiation behavior. METHODS We evaluated various epigenetic neuroectodermal conversion protocols in adult hMSC derived from older donors (>45 versus 18-35 years of age) using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and immunocytochemistry. The protocols included single- and multi-step conversion-differentiation protocols combined with co-culture techniques. Furthermore, the age dependency of mesodermal differentiation potential and cell senescence were investigated. RESULTS The neuroectodermal differentiation potential of hMSC derived from old donors was completely lost, with no cells showing mature neuroectodermal phenotypes using single- and multi-step conversion-differentiation protocols and no improvement of neurogenesis by various co-culture conditions. Comparison of young versus old donor-derived hMSC showed fewer cells expressing early neuroectodermal marker proteins in the latter samples. qRT-PCR showed reduced expression of the proliferation marker KI67 and the neuroectodermal gene NES (nestin) in old donor-derived cells compared with young donor hMSC. Telomere length analysis showed no general cell aging. CONCLUSIONS Our data provide evidence that only young donor-derived hMSC can be epigenetically differentiated in vitro into neuroectodermal cells, pointing towards senescence of multipotentiality of old donor-derived hMSC. There is thus an urgent need to develop better protocols for successful neuroectodermal differentiation of hMSC from old individuals as a prerequisite for autologous cell replacement strategies for neurologic diseases in elderly patients.

Isolation of Neural Crest Derived Chromaffin Progenitors from Adult Adrenal Medulla

Chromaffin cells of the adrenal medulla are neural crest‐derived cells of the sympathoadrenal lineage. Unlike the closely‐related sympathetic neurons, a subpopulation of proliferation‐competent cells exists even in the adult. Here, we describe the isolation, expansion, and in vitro characterization of proliferation‐competent progenitor cells from the bovine adrenal medulla. Similar to neurospheres, these cells, when prevented from adherence to the culture dish, grew in spheres, which we named chromospheres. These chromospheres were devoid of mRNA specific for smooth muscle cells (MYH11) or endothelial cells (PECAM1). During sphere formation, markers for differentiated chromaffin cells, such as phenylethanolamine‐N‐methyl transferase, were downregulated while neural progenitor markers nestin, vimentin, musashi 1, and nerve growth factor receptor, as well as markers of neural crest progenitor cells such as Sox1 and Sox9, were upregulated. Clonal analysis and bromo‐2′‐deoxyuridine‐incorporation analysis demonstrated the self‐renewing capacity of chromosphere cells. Differentiation protocols using NGF and BMP4 or dexamethasone induced neuronal or endocrine differentiation, respectively. Electrophysiological analyses of neural cells derived from chromospheres revealed functional properties of mature nerve cells, such as tetrodotoxin‐sensitive sodium channels and action potentials. Our study provides evidence that proliferation and differentiation competent chromaffin progenitor cells can be isolated from adult adrenal medulla and that these cells might harbor the potential for the treatment of neurodegenerative diseases, such as Parkinsons disease. STEM CELLS 2009;27:2602–2613

Genetic instability and diminished differentiation capacity in long-term cultured mouse neurosphere cells

The potential use of neural stem cells in basic research, drug testing and for development of therapeutic strategies requires large scale in vitro amplification, increasing the probability of genetic instability and transformation. Little is known, however, about potential correlations between long-term culture of neural stem and progenitor cells (NSPCs), changed differentiation and self-renewal capacities, and the occurrence of chromosomal instability. This study investigates the effect of extended culture time on self-renewal, differentiation capacity, cell cycle phase distribution, telomere length, telomerase activity and chromosomal stability on fetal brain-derived cells that form floating sphere colonies (neurospheres). We observed that increased sphere-forming capacity indicative of increased proliferation was accompanied by a decreased ability to differentiate into neural lineages. The high mobility group A (Hmga2) gene positively regulates self-renewal via repression of p16(Ink4a) and p19(ARF) gene expression. This study discerned an upregulation of Hmga2 gene and protein expression and decreased p16(Ink4a) and p19(ARF) gene expression, suggesting that Hmga2 might promote the proliferation of neurosphere cells in long-term culture. Further, our analyses revealed a significant decrease in telomere length after 4 weeks of culturing that is paralleled by a moderate upregulation of telomerase activity. Importantly, regular gain of chromosome 1 with random structural chromosomal aberrations was observed within 16 weeks of neurosphere cell culture. Genetic instability and diminished differentiation capacity seem to be a consequence of long-term culture of neurosphere cells. These data indicate the necessity to analyze self-renewal, differentiation capacity, telomere length, tumor suppressor genes and chromosomal stability in neurosphere cultures prior to their usage in basic research, drug testing or the development of therapeutic strategies.

Isolation, Characterization, and Differentiation of Progenitor Cells from Human Adult Adrenal Medulla

Chromaffin cells, sympathetic neurons of the dorsal ganglia, and the intermediate small intensely fluorescent cells derive from a common neural crest progenitor cell. Contrary to the closely related sympathetic nervous system, within the adult adrenal medulla a subpopulation of undifferentiated progenitor cells persists, and recently, we established a method to isolate and differentiate these progenitor cells from adult bovine adrenals. However, no studies have elucidated the existence of adrenal progenitor cells within the human adrenal medulla. Here we describe the isolation, characterization, and differentiation of chromaffin progenitor cells obtained from adult human adrenals. Human chromaffin progenitor cells were cultured in low‐attachment conditions for 10–12 days as free‐floating spheres in the presence of fibroblast growth factor‐2 (FGF‐2) and epidermal growth factor. These primary human chromosphere cultures were characterized by the expression of several progenitor markers, including nestin, CD133, Notch1, nerve growth factor receptor, Snai2, Sox9, Sox10, Phox2b, and Ascl1 on the molecular level and of Sox9 on the immunohistochemical level. In opposition, phenylethanolamine N‐methyltransferase (PNMT), a marker for differentiated chromaffin cells, significantly decreased after 12 days in culture. Moreover, when plated on poly‐l‐lysine/laminin‐coated slides in the presence of FGF‐2, human chromaffin progenitor cells were able to differentiate into two distinct neuron‐like cell types, tyrosine hydroxylase (TH)+/β‐3‐tubulin+ cells and TH−/β‐3‐tubulin+ cells, and into chromaffin cells (TH+/PNMT+). This study demonstrates the presence of progenitor cells in the human adrenal medulla and reveals their potential use in regenerative medicine, especially in the treatment of neuroendocrine and neurodegenerative diseases.

Chromaffin cells: the peripheral brain

Chromaffin cells probably are the most intensively studied of the neural crest derivates. They are closely related to the nervous system, share with neurons some fundamental mechanisms and thus were the ideal model to study the basic mechanisms of neurobiology for many years. The lessons we have learned from chromaffin cell biology as a peripheral model for the brain and brain diseases pertain more than ever to the cutting edge research in neurobiology. Here, we highlight how studying this cell model can help unravel the basic mechanisms of cell renewal and regeneration both in the central nervous system (CNS) and neuroendocrine tissue and also can help in designing new strategies for regenerative therapies of the CNS.

Differentiation of Chromaffin Progenitor Cells to Dopaminergic Neurons

The differentiation of dopamine-producing neurons from chromaffin progenitors might represent a new valuable source for replacement therapies in Parkinsons disease. However, characterization of their differentiation potential is an important prerequisite for efficient engraftment. Based on our previous studies on isolation and characterization of chromaffin progenitors from adult adrenals, this study investigates their potential to produce dopaminergic neurons and means to enhance their dopaminergic differentiation. Chromaffin progenitors grown in sphere culture showed an increased expression of nestin and Mash1, indicating an increase of the progenitor subset. Proneurogenic culture conditions induced the differentiation into neurons positive for neural markers β-III-tubulin, MAP2, and TH accompanied by a decrease of Mash1 and nestin. Furthermore, Notch2 expression decreased concomitantly with a downregulation of downstream effectors Hes1 and Hes5 responsible for self-renewal and proliferation maintenance of progenitor cells. Chromaffin progenitor-derived neurons secreted dopamine upon stimulation by potassium. Strikingly, treatment of differentiating cells with retinoic and ascorbic acid resulted in a twofold increase of dopamine secretion while norepinephrine and epinephrine were decreased. Initiation of dopamine synthesis and neural maturation is controlled by Pitx3 and Nurr1. Both Pitx3 and Nurr1 were identified in differentiating chromaffin progenitors. Along with the gained dopaminergic function, electrophysiology revealed features of mature neurons, such as sodium channels and the capability to fire multiple action potentials. In summary, this study elucidates the capacity of chromaffin progenitor cells to generate functional dopaminergic neurons, indicating their potential use in cell replacement therapies.

Chromaffin Progenitor Cells from the Adrenal Medulla

Chromaffin cells of the adrenal medulla are neural crest-derived cells of the sympathoadrenal lineage. Different lines of evidence suggest the existence of a subpopulation of proliferation-competent progenitor cells even in the adult state. The identification of sympathoadrenal progenitors in the adrenal would greatly enhance the understanding of adrenal physiology and their potential role in adrenal pathogenesis. Isolation and differentiation of these progenitor cells in culture would provide a tool to understand their development in vitro. Furthermore, due to the close relation to sympathetic neurons, these cells might provide an expandable source of cells for cell therapy in the treatment of neurodegenerative diseases. We therefore aim to establish protocols for the efficient isolation, enrichment and differentiation of chromaffin progenitor cells to dopaminergic neurons in culture.

Valproic acid enhances neuronal differentiation of sympathoadrenal progenitor cells.

The antiepileptic drug valproic acid (VPA) has been shown to influence the neural differentiation and neurite outgrowth of neural stem cells. Sympathoadrenal progenitor cells share properties with neural stem cells and are considered a potential cell source in the treatment of neurodegenerative diseases. The present study therefore aims at modulating the neural differentiation potential of these cells by treatment with the histone deacetylase inhibitor VPA. We studied the epigenetic effects of VPA in two culture conditions: suspension conditions aimed to expand adrenomedullary sympathoadrenal progenitors within free-floating chromospheres and adherent cell cultures optimized to derive neurons. Treatment of chromospheres with VPA may launch neuronal differentiation mechanisms and improve their neurogenic potential upon transplantation. However, also transplantation of differentiated functional neurons could be beneficial. Treating chromospheres for 7 days with clinically relevant concentrations of VPA (2 mm) revealed a decrease of neural progenitor markers Nestin, Notch2 and Sox10. Furthermore, VPA initiated catecholaminergic neuronal differentiation indicated by upregulation of the neuronal marker β-III-tubulin, the dopaminergic transcription factor Pitx3 and the catecholaminergic enzymes TH and GTPCH. In adherent neural differentiation conditions, VPA treatment improved the differentiation of sympathoadrenal progenitor cells into catecholaminergic neurons with significantly elevated levels of nor- and epinephrine. In conclusion, similar to neural stem cells, VPA launches differentiation mechanisms in sympathoadrenal progenitor cells that result in increased generation of functional neurons. Thus, data from this study will be relevant to the potential use of chromaffin progenitors in transplantation therapies of neurodegenerative diseases.

A Defined, Controlled Culture System for Primary Bovine Chromaffin Progenitors Reveals Novel Biomarkers and Modulators

We present a method to efficiently culture primary chromaffin progenitors from the adult bovine adrenal medulla in a defined, serum‐free monolayer system. Tissue is dissociated and plated for expansion under support by the mitogen basic fibroblast growth factor (bFGF). The cultures, although not homogenous, contain a subpopulation of cells expressing the neural stem cell marker Hes3 that also propagate. In addition, Hes3 is also expressed in the adult adrenal medulla from where the tissue is taken. Differentiation is induced by bFGF withdrawal and switching to Neurobasal medium containing B27. Following differentiation, Hes3 expression is lost, and cells acquire morphologies and biomarker expression patterns of chromaffin cells and dopaminergic neurons. We tested the effect of different treatments that we previously showed regulate Hes3 expression and cell number in cultures of fetal and adult rodent neural stem cells. Treatment of the cultures with a combination of Delta4, Angiopoietin2, and a Janus kinase inhibitor increases cell number during the expansion phase without significantly affecting catecholamine content levels. Treatment with cholera toxin does not significantly affect cell number but reduces the ratio of epinephrine to norepinephrine content and increases the dopamine content relative to total catecholamines. These data suggest that this defined culture system can be used for target identification in drug discovery programs and that the transcription factor Hes3 may serve as a new biomarker of putative adrenomedullary chromaffin progenitor cells.

Is there a role for chromaffin progenitor cells in neurodegenerative diseases

Transplantation of cells from the sympathoadrenal lineage has been suggested in the treatment of neurodegenerative diseases and pain. Currently, this approach is not practical due to a shortage of organ donors, lack of tissue homogeneity and expandable cells, and disappointing survival rates of grafted cells. This article suggests that the isolation, propagation and differentiation of chromaffin progenitors from the adult adrenal medulla may be a novel strategy with a powerful therapeutic potential worth considering for autologous transplantation and cell-based therapy. Chromaffin cells of the adrenal medulla are neuroendocrine cells of the sympathoadrenal lineage of neural crest derivatives and therefore are closely related to sympathetic neurons. They are one of the most intensively studied neural crest derivatives. They contain and secrete a wide range of bioactive substances such as catecholamines, but also neuropeptides, growth hormones, including nerve growth factor and endorphins such as met-enkephalin. In contrast to the closely related sympathetic neurons, cells of the adrenal medulla are able to proliferate throughout life. Owing to the close relation of adrenomedullary chromaffin cells to sympathetic neurons, early postnatal adrenal chromaffin cells can be ‘transdifferentiated’ into neuron-like cells with characteristic neurite outgrowth. The existence of multipotent cells within the adrenal anlagen and the adult adrenal medulla is furthermore indicated by the fact that embryonic and adult adrenal cells from different species, including human, co-express catecholaminergic and neural properties. Due to this close relation to neurons, their plasticity, and the restorative neurotrophic growth hormones secreted from chromaffin cells, autologous intrabrain transplantation of adrenomedullary chromaffin cells has raised early hopes of curing neurodegenerative diseases such as Parkinson’s disease. Indeed, improvement of clinical symptoms after adrenal medulla transplantation in a substantial number of Parkinson’s patients has been described worldwide; about 390 patients received autologous adrenal autotransplants from 1988 to 2001. The beneficial effects, however, were transient and longterm survival and functional efficacy of these grafts were poor, and the clinical improvements disappeared after 1–2 years. Transplantation of fetal dopaminergic neural progenitors from the midbrain of aborted embryos and fetuses is another promising strategy. Several clinical open-label trials with these transplants have proven successful in the treatment of Parkinson’s patients where dopamine release was restored in the striatum and to significantly reverse some of the symptoms of the disorder. This initial success, however, was not supported by the results from two controlled NIH trials. Presently, there are no major ongoing trials using transplantation of fetal neural tissue. In recent years, progress in stem cell research has ignited the hope of curing neurodegenerative diseases by transplanting cells differentiated from neuronal or embryonic stem cells. Neural stem cells can be isolated from either the developing or the adult brain and methods for their propagation and differentiation into neurons, astrocytes and oligodendrocytes are established. However, the generation of functional dopaminergic neurons from these multipotent neural progenitors is difficult and has not yet been achieved. Protocols have recently been developed to differentiate human embryonic stem cells (hESCs) into electrophysiologically active neurons with, for example, functional characteristics of dopaminergic neurons. However, the potential therapy of neurodegenerative diseases with hESCs is not without problems, especially in that the tumorigenic properties of hESCs so far restrict their usefulness in clinical cell transplantation. The need for neural precursors in transplantation procedures brings a new momentum to a chromaffin cell-based strategy. The isolation of fetal chromaffin cells from aborted fetuses is one possibility that is now under investigation and the use of these cells is suggested in pain therapy for spinal cord injury or terminal cancer. Owing to their bipotentiality (neural and endocrine), these cells should also bear the potential for neural differentiation and potentially brain repair. Human fetal adrenal tissue, however, is limited or, for ethical reasons, unavailable. In the search for potential sources for cell-based treatment of Parkinson’s disease, chromaffin cell aggregates of the Zuckerkandl’s organ, an extra-adrenal paraganglion with properties similar to the adrenal medulla, are being tested for their suitability. These transplants induced gradual improvement of functional deficits in Parkinsonian rats. This functional regeneration was attributed less to the replacement of functional neurons as to the chronic trophic action of neurotrophic factors secreted by the grafted cells. Molecular Psychiatry (2009) 14, 2–4 & 2009 Nature Publishing Group All rights reserved 1359-4184/09