Isolation of human mesenchymal stem cells from amnion, chorion, placental decidua and umbilical cord: comparison of four enzymatic protocols
To compare four enzymatic protocols for mesenchymal stem cells (MSCs) isolation from amniotic (A-MSC) and chorionic (C-MSC) membranes, umbilical cord (UC-MSC) and placental decidua (D-MSC) in order to define a robust, practical and low-cost protocol for each tissue.
A-MSCs and UC-MSCs could be isolated from all samples using trypsin/collagenase-based protocols; C-MSCs could be isolated from all samples with collagenase- and trypsin/collagenase-based protocols; D-MSCs were isolated from all samples exclusively with a collagenase-based protocol.
The trypsin-only protocol was least efficient; the collagenase-only protocol was best for C-MSCs and D-MSCs; the combination of trypsin and collagenase was best for UC-MSCs and none of tested protocols was adequate for A-MSCs isolation.
KeywordsAmniotic membrane Chorionic membrane Mesenchymal stem cells Neonatal tissues Placental decidua Umbilical cord
Supplementary Fig. 1—Representative images of collected tissues. After cord blood collection, segments of umbilical cord and placenta were collected (a). The umbilical cord (b), amniotic membrane (c), chorionic membrane and placental decidua (d) were separated. Tissues were thoroughly washed with saline before fragmentation, and the umbilical cord vessels were removed. Amniotic membrane (e), chorionic membrane (f), placental decidua (g) and umbilical cord (h).
Supplementary Fig. 2—Cells morphology of cultures of MCSs obtained from neonatal tissues (P3–P5). (A) A-MSCs: heterogeneous cultures containing fibroblast-like cells and circular/polygonal cells. (B) C-MSCs, (C) D-MSCs and (D) UC-MSCs presented a homogeneous population of fibroblast-like cells. Original magnification 100x.
Supplementary Table 1—Immunophenotype comparison of MSCs (P3-P5) derived from amniotic membrane (A-MSC), chorionic membrane (C-MSC), placental decidua (D-MSC) and umbilical cord (UC-MSC) using different protocols, analyzed by flow cytometry. Undifferentiated cells were marked with monoclonal antibodies. The results are presented by mean percentage ± standard deviation.
Supplementary Fig. 3—A representative example of differentiation capacity (P3-P5) of cells isolated from amniotic membrane (A), chorionic membrane (C), placental decidua (D) and umbilical cord (UC). Considering that the different protocols didn’t affect cell differentiation capacity, only one protocol for each cell source is presented. Cultured cell was exposed to medium for induce osteogenic (stained with Alizarin Red), adipogenic (stained with Oil Red) and chodrogenic (stained with Alcian Blue) differentiation. The magnification is indicated on lower right of each image.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflicts of interest.
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