Harvest of functional mesenchymal stem cells derived from in vivo osteo-organoids

doi:10.12336/biomatertransl.2023.04.006

Biomaterials Translational ›› 2023, Vol. 4 ›› Issue (4): 270-279.doi: 10.12336/biomatertransl.2023.04.006

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Harvest of functional mesenchymal stem cells derived from in vivo osteo-organoids

Shunshu Deng¹^,²^,³, Fuwei Zhu¹^,²^,³, Kai Dai¹^,²^,³^,⁴^,^*(), Jing Wang¹^,³^,⁴^,^*(), Changsheng Liu²^,³^,⁴^,⁵^,^*()

1 State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China
2 Key Laboratory for Ultrafine Materials of the Ministry of Education, East China University of Science and Technology, Shanghai, China
3 Engineering Research Center for Biomedical Materials of the Ministry of Education, East China University of Science and Technology, Shanghai, China
4 Frontiers Science Center for Materiobiology and Dynamic Chemistry, East China University of Science and Technology, Shanghai, China
5 Shanghai University, Shanghai, China

Received:2023-09-21 Revised:2023-11-14 Accepted:2023-11-30 Online:2023-12-27 Published:2023-12-28
Contact: Kai Dai, daikai233@foxmail.com;Jing Wang, biomatwj@163.com;Changsheng Liu, liucs@ecust.edu.cn.

Abstract

Abstract:

Bone marrow-derived mesenchymal stem cells (BM-MSCs) play a crucial role in stem cell therapy and are extensively used in regenerative medicine research. However, current methods for harvesting BM-MSCs present challenges, including a low yield of primary cells, long time of in vitro expansion, and diminished differentiation capability after passaging. Meanwhile mesenchymal stem cells (MSCs) recovered from cell banks also face issues like toxic effects of cryopreservation media. In this study, we provide a detailed protocol for the isolation and evaluation of MSCs derived from in vivo osteo-organoids, presenting an alternative to autologous MSCs. We used recombinant human bone morphogenetic protein 2-loaded gelatin sponge scaffolds to construct in vivo osteo-organoids, which were stable sources of MSCs with large quantity, high purity, and strong stemness. Compared with protocols using bone marrow, our protocol can obtain large numbers of high-purity MSCs in a shorter time (6 days vs. 12 days for obtaining passage 1 MSCs) while maintaining higher stemness. Notably, we found that the in vivo osteo-organoid-derived MSCs exhibited stronger anti-replicative senescence capacity during passage and amplification, compared to BM-MSCs. The use of osteo-organoid-derived MSCs addresses the conflict between the limitations of autologous cells and the risks associated with allogeneic sources in stem cell transplantation. Consequently, our protocol emerges as a superior alternative for both stem cell research and tissue engineering.

Key words: anti-replicative senescence, in vivo osteo-organoid, mesenchymal stem cell, recombinant human bone morphogenetic protein 2, stem cell therapy

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Figures/Tables 8

Figure 1. Illustrations of osteo-organoid cell separation procedures. (A) The mouse body soaked in 75% (v/v) ethanol was placed in biosafety cabinet. (B) After removing skin and muscle, a hindlimb was completely cut off, so that the femur and tibia could be used for extraction of bone marrow-derived mesenchymal stem cells (BM-MSCs). (C) The osteo-organoid was hidden in the swollen part of the muscle (red circle) between femur and calf. (D) Osteo-organoids covered with periosteum-like tissue were separated in a 60-mm cell culture dish. (E) The surface of the blade was convex (red curve) to cut the osteo-organoids. (F) The surface of the blade was concave (red curve) to shred the osteo-organoids.

Table 1. Details of cell culture in different experiments

Experiment	Culture vessel	Volume of medium	Seeding density	Other suggestions
Colony forming unit-fibroblast	6-well plate	2 mL per well	5 × 10⁵ cells per well	Replace α-MEM with DMEM to prepare the complete medium
Purification	100-mm dish	15 mL per dish	1 × 10⁷ cells per dish	Culture under the condition of 5% O₂ atmosphere
Differentiation	6-well plate	2 mL per well	2 × 10⁵ cells per well	Use α-MEM containing 10% fetal bovine serum and 1% penicillin-streptomycin as the medium before differentiation assays
Proliferation	60-mm dish	5 mL per dish	2 × 10⁵ cells per well	Incubate the MSC for 2 hours in a medium containing 50 μM EdU

Table 2. Purification operations are based on the characteristics of MSCs

Characteristics	Operations
Adhesion of MSCs are earlier than other cells from marrow	Wash cells using Dulbecco’s phosphate-buffered saline after seeding overnight
MSCs are easy to be digested and suspended	Time of digestion during the cell passage is not recommended to exceed 2 minutes
Low oxygen pressure increases the proliferation and reduces spontaneous differentiation of MSCs	Expand cells under the hypoxic condition (5% O₂)

Figure 2. Morphology of MSCs and CFU-F assay. (A) Primary cells before (P0-before) and after (P0-after) the first refreshing of the medium, and MSCs at 80% confluence at P0 and P1. Red and blue circles indicate spindle-shaped and round cells. (B) Left: Primary cells from osteo-organoids (constructed with recombinant human bone morphogenetic protein 2-loaded gelatin sponge scaffolds) formed larger colonies than that from bone marrow (native BM). Scale bars: 200 μm. Right: The number of colonies in each well of a standard 6-well plate. Data are expressed as mean ± SD (n = 3). ***P < 0.001 (Student’s t-test). CFU-F: colony forming unit-fibroblast; MSCs: mesenchymal stem cells; P: passage.

Figure 3. Cell surface phenotype and multilineage differentiation of MSCs at P2. (A) Flow cytometry of odMSCs (constructed with recombinant human bone morphogenetic protein 2-loaded gelatin sponge scaffolds; BMP) and bone marrow (native). (B) Alizarin red, oil red O or Alcian blue staining after differentiation under the osteogenic, adipogenic or chondrogenic induction. The osteogenic and adipogenic differentiation of odMSCs were more obvious than that of BM-MSCs. Black triangles indicate calcified nodules and mature adipocytes. Scale bars: 200 μm. BM-MSCs: bone marrow-derived mesenchymal stem cells; MSCs: mesenchymal stem cells; odMSCs: osteo-organoid-derived mesenchymal stem cells; P2: passage 2; Sca-1: stem cell antigen-1.

Figure 4. Upper: Ageing process for MSCs from P0 to P2. Black triangles indicate SA-β-Gal positive cells. Scale bars: 200 μm. Lower: The proportions of SA-β-Gal positive cells. The proportion of cell senescence in the native BM-MSCs group (native) increased with passage, while it remained basically unchanged in the odMSCs (constructed with recombinant human bone morphogenetic protein 2-loaded gelatin sponge scaffolds; BMP). Data are expressed as mean ± SD (n = 3). ***P < 0.001, ****P < 0.0001 (two-way analysis of variance followed by Bonferroni’s multiple comparison test). BM-MSCs: bone marrow-derived mesenchymal stem cells; odMSCs: osteo-organoid-derived mesenchymal stem cells; P: passage; SA-β-Gal: senescence-associated β-galactosidase.

Table 3. Time of mesenchymal stem cell passage from the osteo-organoid vs. native bone marrow

Source	P0	P1	Total
Osteo-organoid	3 days	3 days	6 days
Native bone marrow	8 days	4 days	12 days

Figure 5. Cell proliferation of MSCs at P2. (A, B) Scatter diagrams (A) and content of EdU+ cells (B) by flow cytometric analysis. (C, D) Fluorescence confocal image (blue: DAPI; red: EdU; C) and statistical analysis (D). Scale bars: 200 μm. Data are expressed as mean ± SD (n = 3), and were analyzed by Student’s t-test. BM: native bone marrow-derived mesenchymal stem cells; BMP: osteo-organoid-derived mesenchymal stem cells (constructed with recombinant human bone morphogenetic protein 2-loaded gelatin sponge scaffolds); DAPI: 4′,6-diamidino-2-phenylindole; EdU: 5-ethynyl-2′-deoxyuridine; MSCs: mesenchymal stem cells; ns: not significant; P2: passage 2.

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Harvest of functional mesenchymal stem cells derived from in vivo osteo-organoids

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