Int J Stem Cells.  2018 Jun;11(1):121-130. 10.15283/ijsc18020.

The Role of Microenvironment in Preserving the Potency of Adult Porcine Pulmonary Valve Stem Cells In Vitro

• Chalajour
• Siyahian A
• Hanley FL

• KMID: 2413508
• DOI: http://doi.org/10.15283/ijsc18020

Abstract

BACKGROUND AND OBJECTIVE
The potency of tissue resident stem cells is regulated primarily by inputs from the local microenvironment. Isolation of stem cells through enzymatic digestion of tissue may affect epigenetic regulation of cell fate and performance. Here we employ a non-enzymatic method to harvest and investigate tissue resident stem cells from the adult porcine pulmonary valve.
METHODS AND RESULTS
The presence of c-Kit+ stem cells within the valve tissue was confirmed by immunohistochemistry. An in vitro culture of minced valve leaflets was developed under the standard conditions (37°C with 5% CO2). The viability of the cellular outgrowths was evaluated over the subsequent 12 weeks. Under this culture condition, we identified a population of non-adherent c-Kit+ cells and multiple cellular structures mimicking the phenotype of embryonic stem cells at different stages of development. Formation of multinucleated cells through cell fusion provided an active niche area for homing and interaction of the non-adherent c-Kit+ cells. Expression of pluripotency markers Oct-4 and Nanog was detected in the newly formed multinucleated cells but not in mature colonies. Partial cell fusion was shown by fluorescent live-cell tracking, which confirmed intercellular molecular exchange between donor and recipient cells, resulting in altered cytoplasmic protein expression by the recipient cell.
CONCLUSIONS
These results suggest a role for the microenvironment in decrypting the potential of the valve somatic stem cells in vitro. In addition, our data provide evidence for cell fusion, which may play a critical role in reversing somatic cell fate and spontaneous cellular reprogramming.

Keyword

Heart valve; Stem cell; In vitro cell culture; Cell microenvironment; Niche

MeSH Terms

Adult*
Cell Fusion
Cellular Microenvironment
Cellular Reprogramming
Cellular Structures
Cytoplasm
Digestion
Embryonic Stem Cells
Epigenomics
Heart Valves
Humans
Immunohistochemistry
In Vitro Techniques*
Methods
Phenotype
Pulmonary Valve*
Stem Cells*
Tissue Donors

Figure

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  Fig. 1 Expression and colocalization of stem cell markers in the leaflet (A~J). IHC staining showed the presence of c-Kit+ (A; arrows) and rare Oct-4+ cells (B; arrows) within VECs. Expression of α-SMA was observed in a subset of multinucleated cells (C; arrows) below the endothelial surface of the ventricular side. The positive staining for PCNA showed the proliferation potential of VECs and VICs (D; arrows). The expression of pluripotent stem cell markers SSEA-1 (E), SSEA-4 (F), Tra-1-60 (G), and Tra-1-81 (H) was not detected in the representative tissue sections. Co-expression of c-Kit (red) and e-Nos (green) in VECs (I; arrows) was confirmed by immunofluorescent staining. In addition, a subset of c-Kit+ cells (red) was found in the subendothelial region directly adjacent to the α-SMA+ multinucleated cells (J; green). DAPI (blue) was used for counterstaining of the nuclei.

• 

  Fig. 2 Formation of diverse cellular structures from the leaflet during 12 weeks of tissue culture (A~P). Outgrowth of adherent fibroblast-like cells started a few hours after placing the leaflet tissue in the culture; these fibroblast-like cells stabilized the attachment of the leaflet tissue to the culture dish and provided a feeder layer for non-adherent cells (A). Retraction of the confluent feeder layer created a space (B) for formation of multinucleated syncytium-like structures (C) and a niche area (D). By the second week, bubble-like structures of differing sizes and shapes were detected (E). The phenotype of these structures was similar to those of oocytes, 2-cell embryos, 3-cell embryos (F), 4-cell embryos (G), and morulae (H). Formation of large dense colonies and hollow spheres occurred in the confluent cultures between week 3 and 12 (I). The dense colonies were formed either by recruiting the non-adherent cells to the morula-like structures (J) or by the retraction of the mature niche area (K). Interconnecting bundles were generated between adjacent niche syncytia (L, arrow), a process that often was followed by unification of the two structures to form a larger colony. Phalloidin staining (green) and DAPI nuclear counterstaining (blue) showed the structure of the embryonic-like cells and colonies. Oocyte-like cells exhibited a ring-shaped structure with an elongated and peripheral nucleus (M). Multiple nuclei were located on the periphery of 2-cell-embryo-like structure (N), indicating the possibility that other non-adherent cells would be arranged around these structures. Two-dimensional imaging of one blastula-like structure showed a round aggregation of cells with distinct borders between the cells (O). The unification of two adjacent niche areas was promoted by cytoplasmic fusion (P).

• 

  Fig. 3 Characterization of different types of multinucleated cells in a culture of the leaflet (A~D). Expression of c-Kit was detected in the majority of non-adherent mononuclear and multinuclear cells (A, B; red). Versican was expressed by multinucleated cells and found in the inner cavity of blastula-like colonies, suggesting a potential role for versican in cell aggregation and tissue regeneration (A, B; green arrows). Myofibroblast characteristics of the adherent cells surrounding the niche area were identified by expression of α-SMA (C; green). Expression of pluripotent markers Nanog and Oct-4 were detected in the newly formed niche areas before progressively decreasing during development of multinucleated colonies (C, D; red). Expression of the endothelial cell marker e-Nos in mature colonies showed spontaneous differentiation of pluripotent stem cells into endothelial cells (D; green).

• 

  Fig. 4 Tracing fusion of non-adherent cells using fluorescent dyes of two different colors (A~P). Non-adherent cells were collected and stained (separately) with green or red cell-tracker fluorescent dyes; cells of each color were returned to the originating culture. During the first few days, some of the labeled cells attached to the dish, whereas some other cells remained non-adherent (A~D). Cytoplasmic fusion of green and red labeled cells (yellow in the overlay) was observed in syncytium-like structures (E~H), bubble-like structures (I~L), and multinucleated niche areas (M~P). Migration of labeled cells into the existing and newly formed niche areas was detected (H, L, P; arrows).

• 

  Fig. 5 Differentiation of the non-adherent cells in growth culture media. Non-adherent cells were collected from the primary culture of leaflets and grown in DMEM (A), EGM-2 (B), SMCGM (C), MSCGM (D), or RESGRO (E) medium. During the first week, non-adherent cells exhibited low levels of proliferation in DMEM medium (F) and changes in phenotype (e.g., by adhering to culture dishes) in EGM-2 or SMCGM medium (G, H). The proliferation and adhesion rates were strongly increased in MSCGM (I) or RESGRO (J, K) medium. Formation of multinucleated colonies was widely detected in DMEM or RESGRO medium (L, P) after 3 weeks, whereas proliferation of non-adherent cells and the maturation of the colonies was the evident in MSCGM or RESGRO medium (O, Q). Differentiation of non-adherent cells into cells with endothelial or myofibroblast phenotype was detected in EGM-2 or SMCGM culture, respectively (M, N).

Reference

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