3D microscaffoldsare becoming more and more relevant in regenerative medicine, as they lead to the creation of a structure similar to a physiologicalniche. An example is the nano-engineered Nichoid, a 3D structure in which the cells are able  to  proliferate. In  this  work,we  investigated  the proliferation and stemness properties of Er-NPCswhen grown inside the Nichoid, and their potential therapeutic application in the treatment of Parkinson\u2019s Disease.3D microscaffolds are becoming more and more relevant in regenerative medicine, as they lead to the creation of a structure similar to a physiological niche. An example is the nano-engineered Nichoid, a 3D structure in which the cells are able to proliferate. In this work, we investigated the proliferation and stemness properties of Er-NPCs when grown inside the Nichoid, and their potential therapeutic application in the treatment of Parkinson\u2019s Disease

A.M. Di Giulio

B. Barzaghini

F. Rey

G. Al Haj

G. Cerullo

M.T. Raimondi

R. Osellame

S. Carelli

T. Giallongo

T. Zandrini

English

AIR Universita degli studi di Milano

ESB-ITA Meeting 2019, 30 September – 1 October 2019, Bologna, Italy 1 
Abstract — 3D microscaffolds are becoming more and more 
relevant in regenerative medicine, as they lead to the creation of 
a structure similar to a physiological niche. An example is the 
nano-engineered Nichoid, a 3D structure in which the cells are 
able to proliferate. In this work, we investigated the 
proliferation and stemness properties of Er-NPCs when grown 
inside the Nichoid, and their potential therapeutic application in 
the treatment of Parkinson’s Disease. 
Keywords — Parkinson’s disease, Nichoid, biomechanical 
manipulation, regenerative medicine. 
I. INTRODUCTION 
Stem cells therapy represents a promising strategy for 
replacement therapy in neurodegenerative diseases, such as 
Parkinson’s disease (PD), which is the most common 
neurodegenerative diseases after Alzheimer's disease [1]. In 
2011, we isolated a subclass of murine sub ventricular zone-
derived neural progenitors, named Erythropoietin releasing 
post mortem Neural Precursors Cells (Er-NPCs), which show 
high neuronal differentiation capabilities [2].  Er-NPCs exert 
high neuroprotective and regenerative effects when 
transplanted in both traumatic spinal cord injury and 
Parkinson’s disease mice preclinical models [3]-[7]. The use 
of biomaterials allows the generation of active biophysical 
signals for directing stem cell fate through 3D 
microscaffolds, recreating the physiological niche in which 
stem cells are usually present. In 2013 Raimondi’s group 
applied femtosecond laser two-photon polymerization (2PP) 
to fabricate a three-dimensional microscaffold, named 
Nichoid [8]. The Nichoid shows a good ability to maintain 
and preserve the stemness of primary rodent and human stem 
cells [9], [10]. The aim of this study was to investigate: i) the 
proliferation, differentiation and stemness properties of Er-
NPCs following their cultivation in the Nichoid substrate; ii) 
the therapeutic effect and safety of Er-NPCs cultivated in the 
Nichoid in preclinical experimental model of PD. 
II. MATERIALS AND METHOD 
Nichoids were fabricated by femtosecond laser two-photon 
polymerization (2PP) onto circular glass coverslips using a 
hybrid organic-inorganic SZ2080 photoresist. The laser used 
for 2PP was a Yb:KYW system producing pulses of 300 fs 
duration and 1 MHz repetition rate at a wavelength of 1030 
nm [10]. Er-NPCs where isolated from SVZ of CD1 mice 
and characterized for neuronal markers expression as 
described in Marfia et al. 2011 [2]. Er-NPCs were grown 
inside the Nichoid for 7 days (1x104 cells/cm2), counted and 
characterized by means of immunofluorescence, western blot 
and Real Time PCR analysis. To investigate the Er-NPCs 
growth features inside the Nichoid, cells were plated in 
standard growth medium for 7 days, then counted and 
analysed by immunofluorescence analysis, western blot and 
Real Time PCR analysis. Er-NPCs were transplanted in a 
murine experimental model of PD after a 7 days’ growth 
inside the Nichoid. Dopaminergic degeneration in C57BL/6 
mice was obtained through the administration of MPTP [5]-
[7].  7x104 GFP-positive cells were infused in the mouse left 
striatum by stereotaxic injection.  The effects of transplanted 
cells were determined by means of performance tests aimed 
at detecting specific behavioural improvements. The in vivo 
grafted Er-NPCs’ fate and mechanism of action were 
investigated by immunohistochemistry (Fig.1) [5]-[7]. 
 
 
Figure 1: Experimental plan applied for the therapeutic effect evaluation of 
Nichoid-grown Er-NPCs in a preclinical model of PD. 
III. RESULTS 
Seven days after plating, Er-NPCs grown inside the Nichoid 
show a significantly higher cell viability and proliferation 
than in normal floating culture conditions (Fig. 2). 
 
 
Figure 2: Er-NPCs were plated in NSC medium at the density of 1X104 
cells/cm2 at the plating. The analysis was performed three time for each 
condition. Data are expressed as a mean ± SD. The statistical significance of 
the count performed respect to the control is expressed by an * p<0.05, 
** p<0.01 and ***p<0.001 vs control standard floating conditions. 
Therapeutic effect of neural progenitor cells expanded in the 3D 
nano-engineered Nichoid substrate in a Parkinson’s disease 
preclinical model 
S. Carelli1,2, T. Giallongo1, F. Rey1, B. Barzaghini1,4, G. Al Haj1, T. Zandrini3, G. Cerullo3, R. Osellame3,  
A.M. Di Giulio1,2, M.T. Raimondi4 
1Department of Health Sciences, University of Milan, Milan, Italy,  
2PediatricClinicalResearch Center Fondazione “Romeo ed Enrica Invernizzi”, University of Milano, Milano, Italy.  
3 Istituto di Fotonica e Nanotecnologie (IFN)-CNR and Department of Physics, Politecnico di Milano, Milano, Italy. 
4Department of Chemistry, Materials and Chemical Engineering "Giulio Natta", Politecnico di Milano, Milano, Italy 
ESB-ITA Meeting 2019, 30 September – 1 October 2019, Bologna, Italy 2 
 
After the detachment, cells were analysed by Real Time PCR 
and Western Blot analysis, in order to evaluate the influence 
of the Nichoid on the stemness capabilities of Er-NPCs. All 
the three kind of analysis demonstrated that when Er-NPCs 
are grown inside the Nichoid the expression of stemness 
markers SOX2, OCT4 and NANOG is increased, suggesting 
that the Nichoid induces Er-NPCs pluripotency (Fig. 3).  
 
Figure 3: mRNA levels of staminal marker in Er-NPCs grown in the Nichoid  
respect to control floating conditions. Data are expressed as a mean ± SD. 
The statistical significance of the count performed respect to the control is 
expressed by an * p<0.05 and ** p<0.01 vs control standard floating 
conditions 
 
Furthermore, after being re-plated in floating conditions for 7 
more days, Er-NPCs formed smaller but more abundant 
neurospheres with respect to control, suggesting that they 
have the ability to retain a “memory” of the niche in which 
they were previously grown. The replated cells, analysed by 
immunofluorescence, Real Time-PCR and Western blot, also 
present an increase in pluripotency markers SOX2, OCT4 
and NANOG (Fig. 4). 
Figure 4: mRNA levels of staminal marker in Er-NPCs grown both on 
Nichoid and in standard conditions from 7 days and replated in floating 
standard conditions for 7 more days. Data are expressed as a mean ± SD. 
The statistical significance of the count performed respect to the control 
standard floating conditions is expressed by an * p<0.05 and ** p<0.01. 
 
The therapeutic potential and safety of Nichoid-grown NPCs 
was evaluated by their intrastriatal infusion (7x104 cells) in 
the brain of PD affected mice.  Behavioural performances 
were evaluated with two different tests showing that Nichoid-
grown NPCs promoted the recovery of PD symptoms better 
than NPCs maintained in normal floating conditions (Fig. 5). 
 
Figure 5: Horizontal grid test and Vertical grid test show the recovery of 
function in MPTP animals treated or not with Er-NPCs. A group (n=3) of 
MPTP animals were infused with both Er-NPCs detached from Nichoid and 
from normal floating conditions. Data are expressed as a mean ± SD. The 
statistical significance of the count performed respect to the control is 
expressed by an ** p<0.01 and *** p<0.001 vs control; ° p<0,05 and  
°°° p<0.01 vs MPTP. 
 
IV. DISCUSSION AND CONCLUSION 
 
Er-NPCs demonstrate an increase in pluripotency features 
when grown inside the Nichoid, and they maintain this 
increase when they are grown outside the Nichoid. When 
transplanted in a preclinical model of PD, Er-NPCs from the 
Nichoid are safe and lead to a recovery of function, which is 
more efficient than with Er-NPCs grown in standard floating 
conditions. These results together highlight that the Nichoid 
strongly improve the therapeutic potential of stem cells and 
represent a great promise in the field of regenerative 
medicine applied to neurodegenerative disease. 
ACKNOWLEDGEMENTS 
Financial support was received from “Neurogel-en-Marche” 
Foundation (France) to SC; Fondazione “Romeo and Enrica 
Invernizzi” to AMDG. ERC grant NICHOIDS, G.A. 754467. 
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Therapeutic effect of neural progenitor cells expanded in the 3D nano-engineered Nichoid substrate in a Parkinson&#8217;s disease preclinical model

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Therapeutic effect of neural progenitor cells expanded in the 3D nano-engineered Nichoid substrate in a Parkinson’s disease preclinical model

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AIR Universita degli studi di Milano

Therapeutic effect of neural progenitor cells expanded in the 3D nano-engineered Nichoid substrate in a Parkinson&#8217;s disease preclinical model

Abstract

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Full text

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AIR Universita degli studi di Milano

Therapeutic effect of neural progenitor cells expanded in the 3D nano-engineered Nichoid substrate in a Parkinson’s disease preclinical model