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Transplantation of human ESC-derived mesenchymal stem cell spheroids ameliorates spontaneous osteoarthritis in rhesus macaques - PubMed

  • ️Tue Jan 01 2019

. 2019 Aug 21;9(22):6587-6600.

doi: 10.7150/thno.35391. eCollection 2019.

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Transplantation of human ESC-derived mesenchymal stem cell spheroids ameliorates spontaneous osteoarthritis in rhesus macaques

Bin Jiang et al. Theranostics. 2019.

Abstract

It has been demonstrated that mesenchymal stem cells (MSCs) differentiated from human embryonic stem cells (hESCs), name EMSCs, can treat a variety of autoimmune and inflammatory diseases, with similar efficacies to those achieved with MSCs derived from somatic tissues such as bone marrow (BMSCs). The chance increases even higher for EMSCs, than somatic tissue derived MSCs​, to become a cell drug as the former can be produced in large scale from an unlimited hESC line with easier quality control and less biosafety concern. We have further demonstrated that both human ESCs and EMSCs, after aggregation to form spheroids, can tolerate hypoxic and ambient conditions (AC) for over 4 and 10 days, respectively, without loss of their viability and alteration of their functions. Based on these advantages, we decided to test whether EMSC spheroids, made in large quantity and delivered through a long-term distance at AC, can treat osteoarthritis spontaneously developed in rhesus macaques (M. mulatta) monkeys as well as the allogenic MSCs. Methods: Xenogeneic AC-transported EMSC spheroids or allogenic BMSCs were injected into the articular cavity of both knees of the monkeys at 3 animals per group. Another two macaques were injected the same way with saline as controls. Results: Both EMSCs and BMSCs groups showed significant amelioration indicated by the reduction of swelling joint size and amplification of keen flare angle post-treatment, compared to the control group. Examinations via X-ray and MRI also indicated the decrease of inflammation and osteophyma, and recovery of the synovium and cartilage in both treated groups. No sign of allergy or graft versus host disease was observed in the animals. Conclusion: Our results demonstrate that human EMSC spheroids can prevent the osteoarthtitis progression and ameliorate osteoarthritis in the rhesus macaques as well as allogenic BMSCs, and this study shall help advance the clinical application of EMSCs.

Keywords: AC tranportation; Mesenchymal stem cells; Osteoarthritis; Rhesus macaques.; Spheroids.

© The author(s).

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Conflict of interest statement

Competing Interests: R.-H.X. is a founder of ImStem Biotechnology, Inc., a stem cell company. He declares competing financial interests. The remaining authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1

Characterization of BMSCs derived from a rhesus macaque. (A) Process of BMSCs derivation and morphology of the cells, Scale bars: 100 µm. (B) Flow cytometry assay for cell surface markers on the monkey BMSCs. Controls were set up as in Figure 1. (C) Tri-lineage differentiation of the monkey BMSCs. Scale bars: 50 µm.

Figure 2
Figure 2

Characterization of EMSCs derived from the GFP+ Envy hESCs. (A) Formation of EMSC spheroids in hanging drops, morphology of the spheroids and EMSCs in monolayer after delivery and being re-plated in a Petri dish. Scale bars: 1mm (left) and 400 µm (right). (B) Flow cytometry assay for cell surface markers on EMSCs. The red peak refers to a tested sample, and blue and brown peaks refer to isotype and unstained controls, separately. (C) Tri-lineage differentiation of EMSCs into adipocytes, chondrocytes, and osteocytes. Scale bars: 100 µm.

Figure 3
Figure 3

Viability and chondrogenic differentiation of EMSC spheroids post 7 days of ambient delivery. (A) EMSC spheroids underwent delivery at ambient condition for 7 days maintain high survival rate and fast recovery in dish after arrival in host facility. Scale bars: 1 mm. (B) EMSC spheroids delivered under ambient conditions for 7 days retained capability to directly differentiate to chondrocytes in 3D spheroids in chondrocyte differentiation medium, which were positive for the chondrocyte staining Alcian Blue but negative for the osteocyte staining Alizarin Red. EMSCs cultured in 2D (EMSCNor) were differentiated to chondrocytes and osteocytes in the corresponding medium as positive controls. Scale bars: 200 µm.

Figure 4
Figure 4

Treatment of osteoarthritis monkeys with MSC via intra-articular injection. (A) A scheme for BMSC preparation, ambient transportation of EMSC spheroids and intra-articular injection of the MSC into osteoarthritis. (B) The table of monkeys' information and the experimental treatments were shown below.

Figure 5
Figure 5

MSC-mediated amelioration of rhesus osteoarthritis in width of joint and leg extending. (A) The thickening joint width before treatment were described as 100%. The reduction of the thickening joint width were shown following the timeline of cell treatments. Data of three measurements by two experienced veterinarians on each knee are presented as means ± SD. (B) Timeline of the angle of femur and tibia. The macaques of the control group are R1 and R2. The macaques of the BMSCs treatment group are R3, R4 and R5. The macaques of the EMSCs treatment group are R6, R7, R8. Data are presented as means ± SD. (C) The statistics of the reducing joint width of three monkeys are presented as means ± SE. (D) The maximal extension degree of both knees measured as the angle formed by the femur and tibia of three monkeys. Data are presented as means ± SE. The red line graph represents the left knee and the blue line graph represents the right knee. The statistical differences were analyzed using Student's t-test. The significant differences of BMSCs and control groups are presented as #, P < 0.05; ##, P < 0.01. The significant differences of EMSCs and control groups are presented as *, P < 0.05; **, P < 0.01; ***, P <0.001.

Figure 6
Figure 6

MSC-mediated amelioration of rhesus osteoarthritis per X-ray analysis on 3 month and 9 month. (A) X-ray imaging are shown for each monkey in EMSCs treated group. (B) X-ray imaging are shown for each monkey in BMSCs treated group. The bone hyperplasia (green arrow) and osteophyte formation (red arrow) were reduced. The gap of the joint (yellow arrow) increased for the knees of the monkey who received treatment with either EMSCs or BMSCs treatments. RL: Right lateral; LL: Left lateral; L: Left.

Figure 7
Figure 7

MSC-mediated amelioration of rhesus osteoarthritis per MRI analysis. The exudative patellar ligament (red arrows) decreased, bone hyperplasia (yellow arrows) decreased, and the gap of the joint (red straight line) increased for both knees of the monkeys 9 months after the treatment with EMSCs or BMSCs. R: Right; L: Left

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