Differentiation and migration properties of human foetal umbilical cord perivascular cells: potential for lung repair

Abstract

Mesenchymal stem cells (MSC) have been derived from different cultured human tissues, including bone marrow, adipose tissue, amniotic fluid and umbilical cord blood. Only recently it was suggested that MSC descended from perivascular cells, the latter being defined as CD146⁺ neuro-glial proteoglycan (NG)2⁺ platelet-derived growth factor-Rβ⁺ ALP⁺ CD34⁻ CD45⁻ von Willebrand factor (vWF)⁻ CD144⁻. Herein we studied the properties of perivascular cells from a novel source, the foetal human umbilical cord (HUC) collected from pre-term newborns. By immunohistochemistry and flow cytometry we show that pre-term/foetal HUCs contain more perivascular cells than their full-term counterparts (2.5%versus 0.15%). Moreover, foetal HUC perivascular cells (HUCPC) express the embryonic cell markers specific embryonic antigen-4, Runx1 and Oct-4 and can be cultured over the long term. To further confirm the MSC identity of these cultured perivascular cells, we also showed their expression at different passages of antigens that typify MSC. The multilineage differentiative capacity of HUCPC into osteogenic, adipogenic and myogenic cell lineages was demonstrated in culture. In the perspective of a therapeutic application in chronic lung disease of pre-term newborns, we demonstrated the in vitro ability of HUCPC to migrate towards an alveolar type II cell line damaged with bleomycin, an anti-cancer agent with known pulmonary toxicity. The secretory profile exhibited by foetal HUCPC in the migration assay suggested a paracrine effect that could be exploited in various clinical conditions including lung disorders.

Fig 1

Isolation, culture and immunocytochemical characterization of HUCPC. The dissection under sterile condition of foetal and full-term cords was performed to expose the WJ, the vein and arteries (A). After the in vitro expansion of foetal HUCPC (B) the cells showed a homogeneous morphology (B I, 20×) and form ‘round bottom’ colonies (B II, 40×). (C) The growth rate of foetal HUCPC, calculated as cumulative number of population doublings (CPD), was fastest during weeks 4–10 of expansion. (D) The immunocytochemistry analysis at passage 3 of the culture showed that foetal HUCPC are positive for CD146 (I, II, IV: red; III: green) and co-expressed NG2 (I: green), α-SMA (II: green), PDGF-Rβ (III: red) and SSEA-4 (IV: green). Moreover, the SSEA-4⁺ cells (V: red) expressed also Oct-4 (V: green). All the cells (I–V) were stained with DAPI (blue). Magnification: I–II 40×; III–V 60×. All the slides were analysed using a Video Confocal Microscope (ViCo-Eclipse 80i, Nikon) equipped with a Plan Fluor 40× 1.30 DIC H/N2 Oil objective or with a Plan Apo VC 60×/1.40 oil DIC N2 (Nikon).

Fig 6

Foetal HUCPC migration assay. (A) Rat ATII cells, grown to 80% cell confluence in multiwell, were damaged exposing to bleomycin for 40 hrs. After 24 hrs foetal HUCPC, negative for the pro-surfactant protein C (I) and stained with the red fluorescent dye PKH26, were co-cultured in transwells in the presence of the damaged cell line. During 14 days of co-culture, foetal HUCPC migrate towards the rat ATII layer. Negative control was performed with a non-damaged rat ATII cell line under the same culture conditions. (B) The migration was continuously evaluated with inverted fluorescence microscope to observe the rat ATII cell line before the damage with bleomycin (I), after the damage (II), during the PKH26⁺ foetal HUCPC migration (III: red) and integration (IV: red). The migrated PKH26⁺ foetal HUCPC (V: red) co-expressed the epithelial alveolar marker pro-surfactant protein C (VI: green). Moreover, these cells maintain the expression of the perivascular markers CD146 (VII: green) and NG2 (VIII: green). All the cells (V–VIII) were stained with DAPI (blue).

Fig 2

Localization of HUCPC in sections of foetal and full-term HUC by immunohistochemistry. The presence of HUCPC co-expressing CD146 (red) and α-SMA (green) was also detected around microvessels of foetal HUC (A; magnification 400×). Cells co-expressing CD146 and α-SMA were identified with a low frequency in the peripheral vascular wall of full-term HUC (C; magnification 200×). WJ cells expressing CD105 (red) were much lower in full-term cords (D; magnification 200×) than in the foetal cords sections (B; magnification 200×).

Fig 3

Representative flow cytometry analysis of expanded foetal HUCPC. After three passages of culture the cells (n= 24) were detached with trypsin, washed and stained with directly labelled monoclonal antibodies (pink histograms) or exposed to isotype-matched non-immune directly labelled immunoglobulins (blue histograms). Foetal HUCPC were positive for CD146, PDGF-Rβ, α-SMA and NG2, while were negative for CD45, CD34 and CD56 confirming the main features of perivascular cells. Moreover they expressed the typical mesenchymal markers such as CD90, CD73, CD105, CD44, HLA-ABC and they were negative for CD133, CD144 and HLA-DR.

Fig 4

RT-PCR expression profile of foetal HUCPC. RT-PCR was perfumed with total RNA extracted from expanded foetal HUCPC and the cDNA obtained was used for PCR assays to evaluate the expression of embryonic, myogenic, endothelial and hematopoietic markers. Negative controls (lane 1) were performed with the reaction mix without the cDNA. Positive controls (lane 2) were obtained from the corresponding foetal tissues. The samples of foetal HUCPC used were at different passage of culture (lane 3: passage 3; lane 4: passage 6). Cultured foetal HUCPC were positive for the embryonic markers Runx1 and Oct-4, and for CD146. They were negative for the expression of other embryonic markers (Rex1, Sox-2), myogenic markers (MyoD, Myf5), endothelial markers (CD31, CD34, CD144) and hematopoietic markers (CD45, CD34).

Fig 5

(A) I: Alizarin red staining showing area of mineralization on HUCPC cultured in osteogenic differentiating medium (magnification 20×). (A) II: negative control. Slides were acquired with Nikon Eclipse TS100 equipped with a 20×/040 Ph1 ADL. (B) I: Oil Red O staining showing the accumulation of lipid vacuoles in HUCPC after adipogenic treatment (magnification 40×). (B) II: negative control. Slides were acquired with Nikon Eclipse TS100 equipped with a 40×/0.55 Ph1 ADL. (C) Muscle differentiation after 21 days showed the typical myotubes containing three to five nuclei and co-expressing the tracking marker PKH26 (red) and the human dystrophin (I) (green) and spectrin (II) (green) (magnification 40×). (C) III and IV: negative controls. Slides were analysed using a Video Confocal Microscope (ViCo-Eclipse 80i, Nikon).

Fig 7

ELISA assay was used to investigate the presence of soluble factors during the co-culture of foetal PKH26⁺ HUCPC with damaged (grey) rat ATII cell line and without damage (white). In black the soluble factors released by damaged ATII cell line. Supernatants were collected from six independent experiments at different times during the co-culture (24, 48, 72 hrs) and the evaluated soluble factors were KGF, PDGF-BB and VEGF. Data showed an increase of KGF in the co-culture of HUCPC in the presence of the damage (black) in comparison with non damage (grey). KGF was statistically significant at 48 and 72 hrs (*P < 0.05). The results are expressed as mean ± S.E.