Int J Stem Cells.  2018 Jun;11(1):26-38. 10.15283/ijsc17015.

CD73, CD90, CD105 and Cadherin-11 RT-PCR Screening for Mesenchymal Stem Cells from Cryopreserved Human Cord Tissue

• Pham H
• Tonai R
• Wu M
• Birtolo C
• Chen M

• KMID: 2413498
• DOI: http://doi.org/10.15283/ijsc17015

Abstract

BACKGROUND AND OBJECTIVES
Mesenchymal stem cells (MSCs) are self-renewing, non-specialized cells used clinically in tissue regeneration and sourced from bone marrow, peripheral blood, umbilical cord blood and umbilical cord tissue (UCT). To demonstrate an alternative method for MSC detection, cryopreserved UCT and expanded MSC were screened for MSC markers CD73, CD90, CD105 and CDH-11 by RT-PCR.
METHODS AND RESULTS
Human UCT were washed, sectioned, cryopreserved with 10% DMSO and stored in the vapor phase of liquid nitrogen. Fresh and frozen UCT samples were expanded for MSC. UCT and MSC were processed for RNA and screened for CD73, CD90, CD105 and CDH-11 mRNA by RT-PCR. CD73, CD90 and CD105 were detected by flow cytometry and CDH-11 was detected by Western blotting. Short and long-term frozen UCT shows a loss of mRNA expression for CD73 at 33.2±34.0%, CD90 at 6.2±8.2%, CD105 at 17.7±21.6% and CDH-11 at 30.1±26.7% but was not statistically significant to indicate the deterioration. Expanded MSCs from fresh UCT expressed 0.09±0.07-fold CD73, 0.17±0.11-fold CD90, 0.04±0.06-fold CD105 and 0.14±0.08-fold CDH-11. Expanded MSCs from frozen UCTs expressed 0.09±0.06-fold CD73, 0.13±0.06-fold CD90, 0.04±0.05-fold CD105 and 0.11±0.06-fold CDH-11 and confirmed by flow cytometry and Western blotting.
CONCLUSION
CD73, CD90, CD105 and CDH-11 were detected by RT-PCR in cryopreserved UCT and MSC expansion. CDH-11 appears as a useful single target MSC marker for quick screening.

Keyword

CD73; CD90; CD105; Cadherin-11; Mesenchymal stem cells; Umbilical cord tissue

MeSH Terms

Blotting, Western
Bone Marrow
Dimethyl Sulfoxide
Fetal Blood
Flow Cytometry
Humans*
Mass Screening*
Mesenchymal Stromal Cells*
Methods
Nitrogen
Regeneration
RNA
RNA, Messenger
Umbilical Cord
Dimethyl Sulfoxide
Nitrogen
RNA
RNA, Messenger

Figure

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  Fig. 1 Fresh and Cryopreserved UCT expresses CD73, CD90 and CD105 mRNA. (A) Fresh and (B) cryopreserved UCT from 4 different donors were processed for RNA, converted to cDNA and screened for CD73, CD90 and CD105 by RT-PCR. GAPDH was used as an internal control and data are expressed as mean fold expression against GAPDH±SD from 3 experiments. Representative reactions were resolved on a 1.5% agarose gel and captured on an imager.

• 

  Fig. 2 Fresh and cryopreserved UCT expresses CDH-11 mRNA. (A) Fresh and (B) frozen UCT from 4 different donors were processed for RNA, converted to cDNA and screened for CDH-11 by RT-PCR. GAPDH was used as an internal control and data are expressed as mean fold expression against GAPDH±SD from 3 experiments. Representative reactions were resolved on a 1.5% agarose gel and captured on an imager.

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  Fig. 3 Representative microscopy of expanded MSC from cryopreserved UCT. MSC expansion cultures were propagated from cryopreserved UCT. Representative phase-contrast field of views were captured on a digital microscope.

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  Fig. 4 MSC expansion from fresh and cryopreserved UCT express CD90, CD73 and CD105 mRNA. MSC expanded from (A) fresh and (B) cryopreserved UCT from 2 different donors were processed for RNA, converted to cDNA and screened for CD73, CD90 and CD105 by RT-PCR. GAPDH was used as an internal control and data are expressed as mean fold expression against GAPDH±SD from 3 experiments. Representative reactions were resolved on a 1.5% agarose gel and captured on an imager.

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  Fig. 5 MSC expansion from cryopreserved UCT expresses CD73, CD90 and CD105 cell surface markers. MSC expanded from cryopreserved UCT sections were harvested and screened by flow cytometry for (A) positive CD90, CD73, CD105 and CD44 and checked against (B) negative isotype CD90, CD73, CD105 and CD44. (C) Combined positive CD90, CD73, CD105 and CD44 population for MSC expansion from fresh and frozen are presented as percent mean±SD from n=6.

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  Fig. 6 MSC expansion from cryopreserved UCT expresses CDH-11. MSCs expanded from (A) fresh and (B) cryopreserved UCT sections were processed for RNA, converted to cDNA and screened for CDH-11 by RT-PCR. GAPDH was used as an internal control and data are expressed as mean fold expression against GAPDH±SD from 3 experiments. Representative reactions were resolved on a 1.5% agarose gel and captured on an imager. (C) MSCs were processed for protein, denatured in loading buffer containing SDS, resolved on a 4~20% pre-cast gel and transferred onto nitrocellulose membranes. Membranes were probed with CDH-11 or GAPDH antibodies, visualized by ECL and captured on an imager.

Reference

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