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. 2023 Jan 10;12(2):268.
doi: 10.3390/cells12020268.

Multipotent Mesenchymal Stromal Cells from Porcine Bone Marrow, Implanted under the Kidney Capsule, form an Ectopic Focus Containing Bone, Hematopoietic Stromal Microenvironment, and Muscles

Nataliya Petinati  1 , Irina Shipounova  1 , Natalia Sats  1 , Alena Dorofeeva  1 , Alexandra Sadovskaya  1   2 , Nikolay Kapranov  1 , Yulia Tkachuk  3 , Anatoliy Bondarenko  3 , Margarita Muravskaya  3 , Michail Kotsky  3 , Irina Kaplanskaya  4 , Tamara Vasilieva  5 , Nina Drize  1
Affiliations

Multipotent Mesenchymal Stromal Cells from Porcine Bone Marrow, Implanted under the Kidney Capsule, form an Ectopic Focus Containing Bone, Hematopoietic Stromal Microenvironment, and Muscles

Nataliya Petinati et al. Cells. .

Abstract

Multipotent mesenchymal stromal cells (MSCs) are an object of intense investigation due to their therapeutic potential. MSCs have been well studied in vitro, while their fate after implantation in vivo has been poorly analyzed. We studied the properties of MSCs from the bone marrow (BM-MSC) before and after implantation under the renal capsule using a mini pig model. Autologous BM-MSCs were implanted under the kidney capsule. After 2.5 months, ectopic foci containing bones, foci of ectopic hematopoiesis, bone marrow stromal cells and muscle cells formed. Small pieces of the implant were cultivated as a whole. The cells that migrated out from these implants were cultured, cloned, analyzed and were proven to meet the most of criteria for MSCs, therefore, they are designated as MSCs from the implant-IM-MSCs. The IM-MSC population demonstrated high proliferative potential, similar to BM-MSCs. IM-MSC clones did not respond to adipogenic differentiation inductors: 33% of clones did not differentiate, and 67% differentiated toward an osteogenic lineage. The BM-MSCs revealed functional heterogeneity after implantation under the renal capsule. The BM-MSC population consists of mesenchymal precursor cells of various degrees of differentiation, including stem cells. These newly discovered properties of mini pig BM-MSCs reveal new possibilities in terms of their manipulation.

Keywords: differentiation; ectopic foci; implantation; multipotent mesenchymal stromal cells (MSCs); proliferation.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Proliferative and differentiating potential of porcine BM-derived MSCs (BM-MSCs). (A) Cumulative cell production. (B) MSC Growth Kinetics for 15 Passages. Mean values from four independent samples of BM-MSCs obtained from different animals are presented. (C) Control cells stained with Oil Red O. Photo of Pig#106 cells. (D) Induction of adipogenic differentiation stained with Oil Red O. Photo of Pig#106 cells. Black arrows point to fat droplets. Magnification 10×. (E) Control cells stained with Alizarin Red. Photo of Pig#106 cells. (F) Induction of osteogenic differentiation stained with Alizarin red. Photo of Pig#106 cells. Black arrows show calcium deposits. Magnification 10×.
Figure 2
Figure 2
Photos of ectopic foci formed under the kidney capsule of mini pigs after BM-MSCs implantation. (A) Overview of the implant. The diameter of the Petri dish is 10 cm. The implant is approximately 2 × 3 cm. Bones are palpable inside. (Pig#103). (B) Histological sections of implants. As a control, normal kidney with a connective tissue capsule without an implant is represented. Mallory staining. (Pig#107) Red arrow points to the normal renal capsule. Black arrows indicate renal glomeruli. (C) The marginal area of the implant with connective tissue and blood vessels. Mallory staining. (Pig#103) Black arrow points to the renal capsule with part of implant. (D) Ossicles (black arrows) and ectopic hematopoietic foci (red arrows) containing stromal cells. Hematoxylin–eosin staining. (Pig#106). (E) Ossicle. Mallory staining. (Pig#103). (F) Area containing muscle cells. Hematoxylin–eosin staining. (Pig#103). (G) Ectopic hematopoietic foci. The area adjacent to the one presented in (D) is shown. Hematoxylin–eosin staining. (Pig#106). Magnification 10×.
Figure 3
Figure 3
Characteristics of cells that have migrated out of the foci formed under the renal capsule after BM-MSCs implantation. (A) Pieces of focus tissue in the wells of a 6-well plate (Pig#99). The approximate size of the pieces was 0.3 × 0.3 cm. (B) Morphological heterogeneity of cells that migrated out of the implant (Pig#103). (C) Gel electrophoresis of GFP-specific PCR products. DNA of cells that have migrated out of different pieces of the implant was analyzed. (D) Cell morphology of various IM-MSC clones (Pig#106). (E) Control IM-MSCs stained with Oil Red O. (F) IM-MSCs induced to adipogenic differentiation stained with Oil Red O. (G) Control IM-MSCs stained with Alizarin red. (H) IM-MSCs induced to osteogenic differentiation stained with Alizarin red. Magnification 10×.
Figure 4
Figure 4
Characteristics of IM-MSCs. (A) Comparison of clonal efficiency of BM-MSCs and IM-MSCs. BM-MSCs and IM-MSCs were cloned 1 cell per well of the 96-well plate. Cloning efficiency was determined by the Poisson formula. (B) Population doubling time of BM-MSCs and IM-MSCs. (C) The number of performed mitoses. Variability of individual clones from different pigs. (D) Range of population doubling time in IM-MSCs clones from different mini pigs. (E) Individual differences in the proliferative potential of IM-MSCs clones from different mini pigs. (F) Comparison of C-MYC, NES, KLF4 and OCT4 relative gene expression in BM-MSCs and IM-MSCs. Data are presented as Turkey style box plots (A,F), scatter plots (D,E) and bar graphs (B,C). Green circle and purple triangle indicate outliers in corresponding groups.
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