The therapeutic potential of human multipotent mesenchymal stromal cells combined with pharmacologically active microcarriers transplanted in hemi-parkinsonian rats
Introduction
Parkinson’s disease (PD), mainly resulting from the degeneration of the nigrostriatal dopaminergic system, is a progressive neurodegenerative disorder that affects 2% of the population over 65 years of age. Currently the most efficient therapeutic treatment, l-DOPA, aims at replenishing the amount of dopamine missing in the striatum. However, this strategy slowly becomes less effective after long-term treatment and shows undesirable side effects [1], [2]. Cell therapy is an alternative strategy to treat PD and many clinical studies using foetal dopaminergic cells or dopamine-producing cells, such as adrenal chromaffin cells and human retinal pigment epithelium have been performed [3], [4], [5]. These studies gave encouraging results that have provided the proof of principle for cell therapy in PD. However, the outcome was also highly variable between patients and the foetal grafts raised ethical and practical concerns [6], with a poor survival after transplantation [7], [8], [9], [10].
Stem cells, that can self renew and further differentiate into dopaminergic precursors are currently the most studied candidates for cell therapy in PD. However, due to the difficulties in obtaining neural stem cells from adults and the inherent ethical problems to the use of foetal cells or of embryonic stem cells, multipotent mesenchymal stromal cells (MSCs), may represent an alternative cell source to repair the nervous system [5]. Indeed, as they are easily isolated from the bone marrow, autologous grafts can be performed avoiding ethical and availability concerns. MSCs may differentiate into progeny of the three embryonic layers in vitro, including neuronal-like cells, under the influence of matrix molecules and growth factors [11], [12], [13], [14]. Using appropriate driving cues, these cells that may partially originate from the neural crest [15], can be further directed toward a dopaminergic phenotype [16], [17], [18]. Recently, mesenchymal stem-like cells from endometrial origin have also been described as an interesting source of cells for PD therapy due to their ability to produce TH and restore dopamine level in parkinsonian mice [19]. In addition to their neuronal differentiation potential, MSCs possess immunomodulatory properties [20], [21] and have the ability to migrate toward sources of lesions in the brain [22], [23], [24], [25], [26]. Furthermore, some studies showed a functional improvement in the rotational behavior of hemi-parkinsonian rats upon transplantation of rat or human MSCs [27], [28], [29], [30]. As only a very small number of neuronal-like cells were observed in the brain [29], these effects were mostly attributed to the ability of MSCs to secrete various growth factors that protected the degenerating neuronal fibres. These reports encourage further studies with MSCs for cell therapy of PD, but also highlight the need to enhance MSC cell engraftment.
Tissue engineering, which combines cells with a supportive scaffold providing a 3D structure, may help to improve cell engraftment after transplantation [5], [31]. In this way, microcarriers transporting foetal ventral mesencephalic (FVM) cells or adrenal chromaffin cells improved their long-term survival after intracerebral transplantation in hemi-parkinsonian rats [32], [33], [34]. A clinical trial has also reported the safety and efficacy of gelatine microcarriers conveying human retinal pigment epithelial cells for the treatment of PD [35], [36]. Scaffolds providing a biomimetic surface of different extracellular matrix (ECM) molecules or their derived peptides, that stimulate cell survival and differentiation, may further enhance cell engraftment [5], [37]. In this regard, various studies report that laminin (LM) enhances neurite outgrowth of neurons [38] as well as neural stem cell proliferation [39]. In addition, LM may also improve the integration of transplanted cells and their tissue regeneration potential in an ex vivo model of Parkinson’s disease [40]. Finally, this ECM molecule is known to affect stem cell motility [41] and differentiation of MSCs toward a neuronal phenotype, in terms of morphology [42] but also of marker expression [43].
Another way to improve the efficiency of cell grafts is to engineer a scaffold that not only provides a biomimetic surface but also delivers a relevant bioactive growth factor that is released in a controlled manner, further affecting the fate of both transplanted and host cells (see for review [5], [31], [37]). Indeed, synergistic effects between adhesion and growth factor signals to guide and enhance cell differentiation have now been described [44]. In this sense, we have developed an adaptable and efficient device for tissue engineering, the pharmacologically active microcarriers (PAMs). They are biodegradable and non-cytotoxic polymeric microcarriers made of poly(lactic-co-glycolic acid) (PLGA), that with a functionalized surface provide an adequate 3D support for cell culture and/or for their administration. Their microcarrier role, the biomimetic surface and the programmed delivery of an appropriate therapeutic factor may act synergistically to induce and further maintain the survival and/or differentiation of the transplanted cells and their microenvironment, therefore enhancing their engraftment after complete degradation of the vector [45], [46]. The efficacy of this tool was previously demonstrated in a rat PD paradigm using PAMs conveying PC12 cells and releasing nerve growth factor [47], but also with FVM cells attached to PAMs releasing glial cell line-derived neurotrophic factor [48]. In both cases, the PAMs stimulated cell survival and differentiation leading to an improved behavior of the animals.
The main goal of this study was to improve MSC survival, differentiation and tissue repair function after implantation in the striatum of hemi-parkinsonian rats, using PAMs tailored for this application. We chose to work with a homogeneous and well characterized subpopulation of human MSCs that express pluripotent stem cells markers. These cells termed “marrow-isolated adult multilineage inducible” (MIAMI) cells, may generate mature cells derived from all three embryonic germ layers [49], [50]. EGF is now considered as an important factor able to enhance the therapeutic potential of MSCs (see for review Tamama et al. [51].). We recently demonstrated that exposing MIAMI cells to an EGF and bFGF pre-treatment enhances their neural specification and response to neuronal commitment [11]. MIAMI cells further differentiate toward the neuronal lineage on a fibronectin surface in an NT3-dependent manner [16] and this molecule increases β3-Tubulin expression by MIAMI cells [11] and MSCs [52] during in vitro neuronal differentiation. These results appoint NT3 as an ideal protein to encapsulate into PAMs and to use in combination with EGF-bFGF pre-treated MIAMI cells induced toward a dopaminergic phenotype. In order to choose the appropriate biomimetic surface for PAMs, we first screened the panel of integrins expressed by MIAMI cells by RT-qPCR and flow cytometry. We then studied the effect of LM, compared to fibronectin (FN), on the in vitro neuronal differentiation potential of MIAMI cells in terms of cell proliferation, cell length and expression of neuronal markers. Based on these results, PAMs with a biomimetic surface of LM and poly-d-lysine (PDL) were formulated and their total charge as well as the homogeneity of the LM biomimetic surface was characterized by zetametry and immunofluorescence imaging. PAMs delivering NT3 were formulated and the NT3 release kinetics characterized in vitro. These combined properties should act together to stimulate the survival and differentiation of the grafted MIAMI cells toward a neuronal phenotype. After adhesion of MIAMI cells to PAMs, these complexes were first characterized in vitro. Next, using a rat partial progressive 6-OHDA model of PD their effects on MIAMI cell survival and differentiation as well as on the motor behavior of the animals and tissue repair/protection were further evaluated.
Section snippets
Bone marrow harvesting, selection & expansion of MIAMI cells
Whole bone marrow was obtained from vertebral bodies (T1–L5) of a 3 year old male cadaveric donor following guidelines for informed consent set by the University of Miami School of Medicine Committee on the Use of Human Subjects in Research. As previously described [49], isolated whole bone marrow cells were plated at a constant density of 105 cells/cm2 in DMEM-low glucose, containing 5% foetal bovine serum (FBS) (Hyclone, South Logan, Utah) and antibiotics (AB) pansionredally plated in
MIAMI cells integrin subunit screening
In preliminary experiments we observed a higher number of MIAMI cells if expanded on FN compared to other substrates such as collagen (data not shown). This result, together with the reported neuronal inducing effects of LM, prompted us to screen in MIAMI cells the expression of integrins that may interact with FN or LM. During expansion of MIAMI cells, integrin subunits beta 1, alpha 2, 3, 5, 11 and V were highly expressed when evaluated by RT-qPCR. Subunits beta 3, 4 and alpha 1, 4, 6–9 were
Discussion
Adult cells may be easily isolated from the patient body, in particular from accessible tissues (i.e., blood, skin, bone marrow), therefore permitting autologous grafts to be performed in the clinic without ethical problems. For this reason, as well as for their immunomodulatory and tissue repair capacities, their ability to differentiate into neuronal-like cells and to secrete a variety of molecules, the potential of MSCs to treat neurodegenerative disorders, and especially PD, has been
Conclusion
To conclude, PAMs-NT3 transporting MIAMI cells induced a strong functional recovery in rat models of PD, mainly via an improved survival and differentiation of grafted cells. Moreover, the secretion pattern of relevant neuroprotecting/repairing factors by MIAMI cells was positively modified upon combination with PAMs. These factors, secreted by surviving cells, may be responsible for the neuroprotection/repair of the nigrostriatal pathway observed, while a possible secretion of dopamine by
Disclosure of interests
There is no disclosure of interest in this publication.
Acknowledgments
We thank the SCIAM (“Service Commun d’Imagerie et d’Analyse Microscopique”) of Angers for confocal microscopy images as well as the SCCAN (“Service Commun de Cytométrie et d’Analyse Nucléotidique”) of Angers for the use of PCR facilities. We are also thankful to Marie-Claire Venier and François Hindré (INSERM U646, Angers) for their precious scientific advices as well as to Kevin Curtis (Miami Miller School of Medicine, FL) for RT-qPCR analysis of dopaminergic markers.
Grant information: This
References (69)
- et al.
Motor fluctuations in levodopa treated Parkinsonian rats: relation to lesion extent and treatment duration
Brain Res
(1994) - et al.
Adult cell therapy for brain neuronal damages and the role of tissue engineering
Biomaterials
(2010) - et al.
Prospects of stem cell therapy for replacing dopamine neurons in Parkinson’s disease
Trends Pharmacol Sci
(2009) - et al.
Survival of intracerebrally grafted rat dopamine neurons previously cultured in vitro
Neurosci Lett
(1985) - et al.
EGF and bFGF pre-treatment enhances neural specification and the response to neuronal commitment of MIAMI cells
Differentiation
(2010) - et al.
Brain as the Sea of Marrow
Exp Neurol
(2003) - et al.
Development of mesenchymal stem cells partially originate from the neural crest
Biochem Biophys Res Commun
(2009) - et al.
Neurotrophin-directed differentiation of human adult marrow stromal cells to dopaminergic-like neurons
Bone
(2007) Immunomodulatory effects of fetal and adult mesenchymal stem cells
Cytotherapy
(2003)- et al.
Mesenchymal and neural stem cells labeled with HEDP-coated SPIO nanoparticles: in vitro characterization and migration potential in rat brain
Brain Res
(2009)
In vivo tracking of stem cells in brain and spinal cord injury
Prog Brain Res
Increased survival and migration of engrafted mesenchymal bone marrow stem cells in 6-hydroxydopamine-lesioned rodents
Neurosci Lett
Partial recovery of dopaminergic pathway after graft of adult mesenchymal stem cells in a rat model of Parkinson’s disease
Neurochem Int
Regenerative effect of neural-induced human mesenchymal stromal cells in rat models of Parkinson’s disease
Cytotherapy
Autologous transplants of Adipose-Derived Adult Stromal (ADAS) cells afford dopaminergic neuroprotection in a model of Parkinson’s disease
Exp Neurol
Microcarrier enhanced survival of human and rat fetal ventral mesencephalon cells implanted in the rat striatum
Cell Transplant
Intrastriatal transplantation of rat adrenal chromaffin cells seeded on microcarrier beads promote long-term functional recovery in hemiparkinsonian rats
Exp Neurol
Adrenal chromaffin cells on microcarriers exhibit enhanced long-term functional effects when implanted into the mammalian brain
Neuroscience
Spheramine for treatment of Parkinson’s disease
Neurotherapeutics
A method for a more complete in vitro Parkinson’s model: slice culture bioassay for modeling maintenance and repair of the nigrostriatal circuit
J Neurosci Methods
Extracellular matrix effects on neurosphere cell motility
Exp Neurol
Improving the expansion and neuronal differentiation of mesenchymal stem cells through culture surface modification
Biomaterials
Comparison of standard surface chemistries for culturing mesenchymal stem cells prior to neural differentiation
Biomaterials
Combinatorial protein display for the cell-based screening of biomaterials that direct neural stem cell differentiation
Biomaterials
Pharmacologically active microcarriers: a tool for cell therapy
Biomaterials
The role of pharmacologically active microcarriers releasing TGF-beta3 in cartilage formation in vivo by mesenchymal stem cells
Biomaterials
Pharmacologically active microcarriers releasing glial cell line - derived neurotrophic factor: survival and differentiation of embryonic dopaminergic neurons after grafting in hemiparkinsonian rats
Biomaterials
Low oxygen tension inhibits osteogenic differentiation and enhances stemness of human MIAMI cells
Bone
Reversible protein precipitation to ensure stability during encapsulation within PLGA microspheres
Eur J Pharm Biopharm
Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphrenylglycoluril
Biochem Biophys Res Commun
Characterization of behavioral and neurodegenerative changes following partial lesions of the nigrostriatal dopamine system induced by intrastriatal 6-hydroxydopamine in the rat
Exp Neurol
The potential of GDNF as a treatment for Parkinson’s disease
Exp Neurol
Effective GDNF brain delivery using microspheres – a promising strategy for Parkinson’s disease
J Control Release
Striatal implantation of GDNF releasing biodegradable microspheres promotes recovery of motor function in a partial model of Parkinson’s disease
Biomaterials
Cited by (111)
The potential of biomaterials for central nervous system cellular repair
2021, Neurochemistry InternationalImpact of the physico-chemical properties of polymeric microspheres functionalized with cell adhesion molecules on the behavior of mesenchymal stromal cells
2021, Materials Science and Engineering CHuman dental stem cells of the apical papilla associated to BDNF-loaded pharmacologically active microcarriers (PAMs) enhance locomotor function after spinal cord injury
2020, International Journal of Pharmaceutics