Expansion of human umbilical cord derived mesenchymal stem cells in regenerative medicine

Table 1 Mesenchymal stem cell specific antibodies used in immunocytochemistry

Primary antibody markers	Molecular function and binding	Working dilution	Manufacturer information
CD73	Hyaluronan receptor	1:100	550256, BD Pharmingen, United States
CD105	Endoglin	1:100	560839, BD Pharmingen, United States
Vimentin	Epithelial-mesenchymal transition	1:100	V6389, Sigma-Aldrich, United States
CD29	Membrane glycoprotein	1:100	MAB-1981, Chemicon International, United States
CD90	Thymocyte antigen 1	1:100	CL005AP, EMD Millipore Corp., United States
Stro-1	Mesenchymal cell precursor marker	1:100	14-6688-82, Molecular Probes, Invitrogen, United States
Lin28	Cell surface MSC marker	1:100	PA1-096, Molecular Probes, Invitrogen, United States
CD45	Lymphocyte antigen	1:100	CBL415, BD Pharmingen, United States
HLA-DR	MHC class II immunogenic marker	1:100	14-9956-82, Molecular Probes, Invitrogen, United States
Secondary antibody	Conjugated label	Working dilution	Manufacturer information
Goat anti-rabbit IgG	Alexa Fluor 546	1:200	A-11010 Molecular Probes, Invitrogen, United States
Goat anti-mouse IgG	Alexa Fluor 546	1:200	A-11030 Molecular Probes, Invitrogen, United States

MHC: Major histocompatibility complex; IgG: Immunoglobulin G.

Table 2 Gene expression primer sequences for quantitative polymerase chain reaction

Marker name	Primer sequence	Annealing temperature (°C)
MSC pluripotency markers
GAPDH	F: 5’-CACCATGGGGAAGGTGAAGG-3’	57.8
	R: 5’-AGCATCGCCCCACTTGATTT-3’
NANOG	F: 5’-AAGGCAAACAACCCACTTCTG-3’	56
	R: 5’-CCAGTTGTTTTTCTGCCACCT-3’
Sox-2	F: 5’-CCCTGCAGTACAACTCCATGAC-3’	57.7
	R: 5’-GGTAGTGCTGGGACATGTGAAG-3’
Oct-4	F: 5’-GATGTCAGGGCTCTTTGTCCA-3’	57.1
	R: 5’-TACTCTCCCCAGCTTGCTTTG-3’
MSC proliferation markers
PAX 6	F: 5’-AGCTCGGTGGTGTCTTTGTC-3’	60
	R: 5’-CACTCCCGCTTATACTGGGC-3’
BMP2	F: 5’-AGCTGGGCCGAGGA-3’	58
	R: 5’-TCGGCTGGCTGCCCT-3’
TGF β1	F: 5’-CAAGGCACAGGGGACCAG-3’	58
	R: 5’-CAGGTTCCTGGTGGGCAG-3’
Senescence marker
HMBS	F: 5’-GGCAATGCGGCTGCAA-3’	57
	R: 5’-GGGTACCCACGCGAATCAC-3’
hTERT	F: 5’-GACGTGGAAGATGAGCGTG-3’	57
	R: 5’-GACGACGTACACACTCATC-3’
Mycoplasma strains
Mycoplasma hominis	F: 5’-CAATGGCTAATGCCGGATACGC-3’	58
	R: 5’-GGTACCGTCAGTCTGCAAT-3’
Mycoplasma arginini	F: 5’-AGAGTTTGATCCTGGCTCAGGA-3’	58
	R: 5’-TCAACCAGGTGTTCTTTCCC-3’
Cytomegalovirus
Human cytomegalovirus IE	F: 5’-CCAAGGCCACGACGTTCCTGCAGACTA-3’	58
	R: 5’-TGCTCCTTGATTCTATGCCGCACCA-3’

GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; Sox-2: Sex-determining region Y-box 2; BMP2: Bone morphogenetic proteins; Oct-4: Octamer-binding transcription factor; PAX6: Paired-box 6; TGFβ1: Transforming growth factor beta1; HMBS: Hydroxymethyl-bilane synthase; hTERT: Human telomerase reverse transcriptase; MSC: Mesenchymal stem cell.

Table 3 Donor’s information and sample inclusion

Donor	Age	Anti-HCV1	Anti-HBV1	SYPHILUS1
1	22	-	-	-
2	25	-	-	-
3	26	-	-	-
4	21	-	-	-
5	27	-	-	-
6	29	-	-	-

¹Non-reactive: Negative (-).

HCV: Hepatitis C virus; HBV: Hepatitis B virus.