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ELECTRONIC SUPPLEMENTARY MATERIAL

Human Umbilical Cord Blood Mesenchymal Stem Cells protect mice brain after trauma


Elisa R ZANIER MD, Mery MONTINARO BS, Mariele VIGANO’ PhD, Pia VILLA PhD, Stefano FUMAGALLI BS, Francesca PISCHIUTTA BS, Luca LONGHI MD, Matteo L LEONI MD, Paolo REBULLA MD, Nino STOCCHETTI MD, Lorenza LAZZARI PhD

and Maria-Grazia DE SIMONI PhD

From the Department of Neuroscience (ERZ, MM, PV, SF, FP, LL, MGDS), Mario Negri Institute for Pharmacological Research; Cell Factory (MV, PR, LL), Center of Transfusion Medicine, Cellular Therapy and Cryobiology, Department of Regenerative Medicine, Milano, Italy; CNR (PV), Intitute of Neuroscience, Milano, Italy and University of Milano (LL, MLL, NS), Department of Anesthesia and Critical Care Medicine, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico Milano, Italy.


1Mario Negri Institute, Milano, 2Italy, Cell Factory, Center of Transfusion Medicine, Cellular Therapy and Cryobiology, Department of Regenerative Medicine, 3CNR, Intitute of Neuroscience, Milano, Italy , 4University of Milano, Department of Anesthesia and Critical Care Medicine, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milano, Italy.
Correspondence to:
Maria-Grazia De Simoni, Ph.D.

Laboratory of Inflammation and Nervous System Diseases

Mario Negri Institute

20156 Milano - Italy

Office #: +390239014505

Fax #: +39023546277



Email: desimoni@marionegri.it

SUPPLEMENTARY MATERIALS

Supplementary Table 1

Supplementary Figure 6



Supplemental Table 1: Primers used for relative real-time polymerase chain reaction analysis of nestin, musashi-1, neurogenin, NRCAM, β-tubulin III, NSE, TRKA, MAP-2, GFAP, hTERT, c-myc, p53, GAPDH, β-2-Microglobulin transcripts on cultured CB-MSC

 

Forward

Reverse

Nestin

5’-CTG GCG CAC CTC AAG ATG T

5’ GAA GTT GGG CTC AGG ACT GG

Musashi-1

5’-GGG TTT CGG CTT CGT CAC TTT CA

5’ –TTT GAG AGT GAG GAC ATC GTGG

Neurogenin

5'-CCA CCG CAT GCA CAA CTT G

5'-CCC ATC TAT TGC CTG CTG ACT A

NRCAM

5'-GGC CAC CAA CAT TTT TAA CTC C

5'-CGC CAT CTA TTT TTC TGC TGG

β-TubulinIII

5'-CAT CAG TGA TGA GCA TGG CAT

5'-CAG TTT TCA CAC TCC TTC CGC

NSE

5'-TGG CTT TGC CCC CAA TAT C

5'-CCA ATC ATC CTG GTC AAA TGG

TRKA

5'-CAA CAG GAA GAA CGT GAC GTG

5'-GTA GTT GCC GTT GTT GAC GTG

MAP2

5'-CCT TTG AGA ACA CGA CAC AAC G

5'-CAC CCT CTT CAT CCT CCC TGT A

GFAP

5'-AAA CCA ACC TGA GGC TGG AAG

5'-CAT ACT GCG TGC GGA TCT CTT

hTERT

5’-CTG GCT GAT GAG TGT GTA

5’-CAA GTG CTG TCT GATT CC

c-myc

5’-ATG TAA ACT GCC TCA AAT

5’-AAT TCT GTG TAA CTG CTA TA

p53

5’ATG TTT GGG AGA TGT AAG AA

5’-GGT TAG TAC GGT GAA GTG

GAPDH

5'-GCT TGT CAT CAA TGG AAA TCC C

5'-TTC ACA CCC ATG ACG AAC ATG

β-2-Microglobulin

5’-CATT CCT GAA GCT GAC AGC ATT C

5’-TGC TGG ATG ACG TGA GTA AAC C


Fig. S1. Tissue sampling for quantification of immunostained area. Field selection was performed using a BX61 Olympus microscope equipped with a motorized stage. A) For CD11b and CD68 quantification fields at 40x magnification were selected at 0.4mm posterior to bregma (12 fields), at 1.6mm posterior to bregma (12 fields) and at 2.8mm posterior to bregma (9 fields). The first raw of fields was positioned at the contusion edge, spacing each field by 361.2μm (distance between centres of the fields). A second and a third raw of fields were positioned further from the lesion and aligned to the first raw. Distance between each raw was 722.4μm. B) For GFAP quantification ten 40x magnification fields were selected at 0.4 mm, 1.6 mm and 2.8 mm posterior to bregma and fields belonging to the scar area (white rectangles) and to peri scar area (black rectangles) were separately quantified. Scar fields were positioned at the edge of the contused cortical tissue separated by 361.2μm, while peri scar fields were positioned aligned to scar fields, at a distance of 722.4μm from them.



Fig. S2. CB-MSC cumulative population doublings and senescence-related genes expression profile. A) Representative curve of the growth rate expressed as cumulative number of population doublings (CPD) during passages. B-D) Quantitative expression of p53, hTERT and c-myc in CB-MSC at passage 3 and 6 relative to MCF7.



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