Gene Technology

A Paradigm Shift in Bone Regeneration Therapy: Using Mesenchymal Stem Cells and the CRISPR-Cas9 Technology

Takashi Narai^1*, Yuji Nakayama^2*, Isamu Kodani¹ and Kenji Kokura^{3 1}Division of Oral and Maxillofacial Surgery, Department of Sensory and Motor Organs, School of Medicine, Tottori University, 36-1 Nishi-Cho, Yonago 683, Japan
²Division of Radioisotope Science, Research Initiative Center, Organization for Research Initiative and Promotion, Tottori University, 86 Nishi-Cho, Yo, Japan
³Department of Biomolecular Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan
^*Correspondence: Dr. Takashi Narai, Division of Oral and Maxillofacial Surgery, Department of Sensory and Motor Organs, School of Medicine, Tottori University, 36-1 Nishi-Cho, Yonago 683, Japan, Email: Dr. Yuji Nakayama, Division of Radioisotope Science, Research Initiative Center, Organization for Research Initiative and Promotion, Tottori University, 86 Nishi-Cho, Yo, Japan, Email:

Received: 21-Dec-2020 Published: 11-Dec-2020

Abbreviations

MSCs: Mesenchymal Stem Cells; ES: Embryonic Stem; iPS: induced Pluripotent Stem; BGLAP: Bone Gamma-Carboxyglutamate Protein; hiMSC: Human Immortalized MSCs; EGFP: Enhanced Green Fluorescence Protein.

Introduction

We demand high-quality bone regeneration treatment for diseases of the oral and maxillofacial region, such as cleft lip and palate [1-3] facial trauma [4-6], and bone reconstruction after tumor resection [7-9]. Facial bones are not only essential for aesthetics but also indispensable for maintaining individuals' dignity, masticatory function, and conversation abilities. Therefore, bone regeneration always should be a reliable and efficient treatment method. The current mainstream approaches to bone regeneration treatment are autologous bone grafting [10] and artificial bone grafting with bone substitute materials [11,12]. However, these ‘grafting’ approaches have not been achieved satisfactory treatment results: There are multiple surgical sites for harvesting donor bone fragments for autologous bone grafting, but this entails unavoidable surgical invasion, and postoperative infection and rejection are likely to occur when using artificial bones. In such bone regeneration treatments, undifferentiated cells, transplanted with or within an appropriate support system, that is, in a suitable microenvironment, are expected to differentiate into osteoblasts. However, it takes time to induce bone differentiation in the graft or implantation site when using current bone regenerative approaches, such as autologous bone grafting. As a result, the bone regenerative ability is insufficient, leading to low treatment success. A Grafting approach with either autologous, artificial bone, or in combination may remain as one of standard treatment options in future. But, to improve current clinical results, elucidation of the bone regeneration mechanism at the cellular level is extremely important for establishing new therapeutic strategies for the highly sought-after bone regenerative medicine.

In recent years, a methodology for enhancing the osteogenic differentiation at the transplantation site by utilizing the bone differentiation ability of Mesenchymal Stem Cells (MSCs) has attracted attention as a solution to the conventional bone regeneration treatment problems. Basic [13-15] and clinical [16-18] research is being actively pursued, aiming to assess the utility of in vitro-cultured MSC transplantation to treat bone defects. Indeed, it is easier and faster to collect MSCs from the living body than it is to collect bone fragments used for autologous bone grafts. The ethical hurdles for clinical application of MSCs are lower than those for Embryonic Stem (ES) cells and induced Pluripotent Stem (iPS) cells. Currently, many of the recent attempts to use MSCs for bone defect treatments focus on origins or properties of cells to be introduced; such as the tissue from which the MSCs are derived, the donor's age, and the cell culture history and so on. These conditions are important for successful optimization in treatment, however, it is still not an critical improvement measures focusing on the differentiation potential of stem cells within. Thus, it is necessary to realize a condition or method to efficiently induce osteogenic differentiation in MSCs not only ‘before’, but ‘after’ transplantation. To this end, it is necessary to establish cellular experimental system, ideally real-time techniques, to track the osteogenic differentiation process of viable MSCs and clarify the molecular mechanism that follows.

Tissue engineering is generally defined as technology which applies the principles of engineering and the life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function [19]. Among general strategies in tissue engineering, an isolation of genetically modified cells with improved function is widely performed in the course of development in regenerative medicine. Historically, genetic modification was mainly performed in ES cells so that researchers could get insight for specific function of modified genes not only through in vitro differentiation experiment, but through in vivo production of genetically modified ES cell derived animals. Due to low efficiency in genetic modification such as homologous DNA recombination in somatic cells, ES cells and its derived animals are valuable. Because either from in vitro differentiation model or from produced animals, genetically modified somatic cells can be isolated. Thus, a method that can genetically modify DNA in somatic cells, especially in human cells, was long been required.

The situation has changed with the advent of transcription activator-like effector nuclease and zinc finger nuclease, technologies that efficiently modify (edit) genomic DNA, even in somatic cells. More decisively, genome editing by CRISPRCas9 (clustered regularly interspaced short palindromic repeats- CRISPR associated protein 9) transformed this field. It was first reported in vitro in 2012 [20]. After that, genome editing results in human cells were reported in succession [21-23]. Accordingly, successful application in mammalian genome editing accelerated cell engineering, and theoretically any kind of cells from soma can be modified including MSCs via CRISPR-Cas9 technology.

We would like to discuss in this review how cell or tissue engineering combined with genome editing technologies such as CRISPR-Cas9 can be utilized or adopted for MSCs application in the field of a bone regeneration therapy. We will also discuss how the results could contribute to creating new bone regeneration therapies and their therapeutic and clinical application potential.

Potential and Limitations of Mscs in Bone Regeneration Therapy

MSCs have a self-renewal ability and can differentiate into various cell types such as osteoblasts, adipocytes, and chondrocytes [24]. MSCs can be collected from multiple sites, including bone marrow, adipose tissue, and cord blood [25]. Thus, as a cell source for bone regenerative medicine, they have the advantage in collection techniques that are less surgically invasive than those used for autologous bone grafts. Furthermore, when using the patient's MSCs, safety issues, such as the risk of tumorigenesis when using genetically manipulated iPSCs or ethical issues, as those associated with ES cells, are lower. Thus, MSCs have many useful aspects when considering them for clinical applications, comparing with using pluripotent stem cells such as ES cells or iPS cells [26].

The collected MSCs can be cultured in vitro in preparation for use in bone regeneration treatment. The required number of cells are determined based on the size of bone defect and the patient’s status. One treatment strategy being attempted is to promote bone regeneration by transplanting the cultured MSCs to their proper place [5,17,18]. Positive results have been obtained when MSCs were administrated with scaffolding materials and/or osteogenic differentiation-inducing factors to treat relatively small bone defects [17,18]. However, these strategies could be insufficiently effective for moderate or large bone defects because large numbers of cultured MSCs are required to fill such defects. Moreover, as long term culture may cause genetic instabilities, to obtain large number of MSCs to be administrated for relatively large bone defect is still challenging issue to be overcome [27]. In addition to the cell number issue described above, self-renewal and differentiation ability of multipotent MSCs may be declined during longer period of culture [28]. MSCs are thought to be heterogeneous population and, indeed, differentiation-induction and proliferative ability differ even with MSCs, depending on the tissue of origin and the donor's state, and a differentiation-inducing protocol suitable for each collected cells is required [29-32].Thus, to utilize MSCs as a source for bone regenerative medicine, it is essential to examine the primary conditions in various stages, such as MSCs collection, culture condition, co-transplanting materials and/or safety management. Additionally, since MSCs are multipotent, it is challenging to predict or control which lineage they would differentiate into or the differentiation efficiently at the transplantation site. A major issue in bone regenerative medicine is that osteogenic differentiation at the bone graft site cannot be controlled and that only part of the MSCs after transplantation seem to undergo bone differentiation. The only way to solve these problems is to dissect the MSCs differentiation process at the molecular level and identify cell-dependent and environment-dependent elements that control it. However, elucidating this information for native MSCs is highly challenging due to the diversity of lineages and not unlimited stabilities even under appropriate culture condition. Therefore, constructing a system that can analyze the cell properties in live cell is highly desirable. To this end, efficient and accurate tissue engineering of MSCs are required including human MSCs.

A Fusion of Mscs And The Crispr-Cas9 Technology in Bone Regeneration Therapy

When considering using MSCs for bone regenerative medicine, one of current problems is the absence of an evaluation method to assess the process of osteogenic differentiation performance of transplanted MSCs and/or osteogenic-directed cells. As discussed above, molecular dissection of MSCs osteogenic differentiation is a key to overcome this issue. Two directions may be considered: Isolation of an absolute osteogenic differentiation marker (s), and isolation of live and pure osteoblast population. Only when these are available will it be possible to search for conditions that efficiently and reliably differentiate MSCs into the osteoblastic lineage. Currently, however, a real-time screening system to find compounds, molecules, or microenvironment that would promote or inhibit osteogenic differentiation has not yet been developed.

Osteogenic differentiation markers isolated to date include alkaline phosphatase, Bone Gamma-Carboxyglutamate Protein (BGLAP), Bone Sialoprotein I (BSP-1), and Dentin Matrix Protein 1 (DMP1). However, these are all endogenous markers, including transcription factors, and monitoring their expression in live cells is challenging. Therefore, the introduction of genetic markers by cell engineering is required. Among the wide variety of genome editing tools so far developed, the advent of the CRISPR-Cas9 technology has evolutionized genome editing strategy for mammalian cell engineering [21-23]. Accordingly, these advantages have led to the development of research related to genome editing in stem cells including human MSCs [33-35]. Attempts have been made to construct a system that could monitor the induction of osteogenic differentiation in living cells using CRISPR-Cas9 by other [35] and by our [36] group recently. In these studies, by introducing fluorescent protein which express during osteogenic differentiated cells, it was possible to monitor osteogenic differentiation induction in viable modified cells by observing fluorescent protein expression. Although next essential steps will be a detailed molecular confirmation of the properties of cells which express these fluorescent markers toward identification of bona fide osteogenic differentiation markers. Different from endogenous markers described above, these markers are preferably cell surface markers, as these will be valuable to develop a system to recover osteogenic cells derived from native, non-genetically modified MSCs in future. Our study used human immortalized MSCs (hiMSCs) to establish osteogenic monitoring cells [36]. One of the main reason for this choice is long term process of cloning. The hiMSCs we used are multi-potent as the original MSCs and, once osteogenic differentiation is induced, express the osteogenic differentiation markers known to date [37,38]. However, in the case of native MSCs, there was a possibility that their properties including osteogenic differentiation potential may be declined in the course of establishing of genetically modified MSCs. Thus, we used hiMSCs and successfully knocked-in the Enhanced Green Fluorescence Protein (EGFP) reporter gene under the promotor of the BGLAP gene, a differentiation marker for mature osteoblasts, by using CRISPRCas9 system. The expression of BGLAP could be monitored by EGFP signals in the established hiMSC lines under live culture, indicating the induction of osteogenic differentiation in these cells. Moreover, we conducted cell sorting experiment to purify EGFP-positive cells to evaluate the relationship between EGFP signal and osteogenic properties. As a result, we demonstrated that BGLAP expression was increased in these EGFP positive population, compared with expression level in EGFP-negative population and with parental (non-genetically modified) hiMSCs. This indicated that EGFP expression reflect osteogenic differentiation induction. Furthermore, we confirmed that EGFP expression were maintained at least 5 weeks of osteogenic differentiation induction (unpublished results) and that from two weeks of induction EGFP positive cells became positive for Arizarin-red S staining, a hallmark of calcification, indicating functionality of this population as osteoblast-directed cells. By using DMP1 gene as knock-in target gene, Fahimeh et al. also reported monitoring osteoblast differentiation in live cells. Although MSCs used were different (hiMSCs vs. native), CRISPR-Cas9 tissue engineering successfully visualized osteogenic differentiation in live cells. These monitoring system and differentiated monitoring cells will be valuable for molecularly dissecting osteoblast differentiation and/or elucidating molecular properties of osteoblast cells by applying an omics analysis in future. In regard to this, we demonstrated that live and pure osteoblasts can be enriched and this would enable us to conduct focused and detailed analysis of the purified osteoblasts. These results cannot contribute to bone regenerative medicine unless they lead to the identification of the osteoblast-specific cell surface markers. And next, these results should be validated using unmodified patient MSCs cells for clinical application, hoping to create an efficient and effective new bone regeneration cell therapy that could replace autologous bone grafting.

Finally, our work may provide a system that can reassess current bone regenerative medicine methodologies. As discussed in this review, basal problem of MSC transplantation for bone regenerative medicine may due to poor or declining osteogenic differentiation efficiency in transplanted site. There are several potential strategies to tackle this challenge. First, prepare undifferentiated MSCs to be transplanted destined for osteogenic differentiation in in vitro culture. Second, prepare or direct a transplant microenvironment in which introduced MSCs are able to differentiate into bone efficiently. Third, transplant osteogenic cells differentiated in in vitro culture system. Although additional components such as artificial substances and/or factors to drive osteogenic induction may be required, manipulating or directing cell fate of MSCs to osteogenic lineage in vitro may be challenging, but, promising prerequisite for successful transplantation. In one reports, good results in human fracture treatments were achieved using osteoblasts obtained by inducing osteogenic differentiation of MSCs in vitro [39]. This suggested that transplantation of purified osteoblast might be give better results in osteogenic differentiation efficiency and treatment outcomes than the case of transplanting MSCs itself directly. Then, if this is true, our established monitoring cells or derivatives in future by other groups will be valuable to evaluate the advantage in using differentiated osteoblast for bone regenerative transplantation.

Conclusion

We recognized the possibility of a paradigm shift in bone regenerative medicine by applying the CRISPR-Cas9 genome editing technology to MSCs. Genome editing technology using CRISPR-Cas9 in particular has realized the production of modified MSCs useful for bone regenerative medicine more efficiently and easily than before. This fact provided a new insight into the process of osteogenic differentiation of MSCs in vitro and isolation of purified osteoblast from native MSCs are necessary to continue developing a bone regenerative medicine. In future, gap between outcome in basic research and requirement in clinical setting should be filled to promote bone regeneration treatment. To this end, genetic modification technologies such as CRISPR-Cas9 system will be powerful solution to bring the gospel of bone regeneration treatment for both doctors and patients.

Conflict of Interest

The authors declare that there are no conflicts of interest.

Acknowledgment

We would like to thank Editage (www.editage.com) for English language editing.

REFERENCES

1. Khalil W, de Musis CR, Volpato LE, Veiga KA, Vieira EM, Aranha AM. Clinical and radiographic assessment of secondary bone graft outcomes in cleft lip and palate patients. Int Sch Res Notices. 2014.
2. Du Y, Zhou W, Pan Y, Tang Y, Wan L, Jiang H. Block iliac bone grafting enhances osseous healing of alveolar reconstruction in older cleft patients: A radiological and histological evaluation. Med Oral Patol Oral Cir Bucal. 2018;23(2):e216-e224.
3. Mossaad A, Badry TE, Abdelrahaman M, Abdelazim A, Ghanem W, Hassan S, et al. Alveolar cleft reconstruction using different grafting techniques. Maced J Med Sci. 2019;7(8):1369-1373.
4. Slusarenko da Silva Y, de Gouveia MM, Alves CA, Migliolo RC. Late treatment of a mandibular gunshot wound. Autops Case Rep. 2015;5(1):53-59.
5. Bajestan MN, Rajan A, Edwards SP, Aronovich S, Cevidanes LHS, Polymeri A, et al. Stem cell therapy for reconstruction of alveolar cleft and trauma defects in adults: A randomized controlled, clinical trial. Clin Implant Dent Relat Res. 2017;19(5):793-801.
6. De Ponte FS, Falzea R, Runci M, Siniscalchi EN, Lauritano F, Bramanti E, et al. Histomorhological and clinical evaluation of maxillary alveolar ridge reconstruction after craniofacial trauma by applying combination of allogeneic and autogenous bone graft. Chin J Traumatol. 2017;20:14-17.
7. Omeje K, Efunkoya A, Amole I, Akhiwu B, Osunde D. A two-year audit of non-vascularized iliac crest bone graft for mandibular reconstruction: technique, experience and challenges. J Korean Assoc Oral Maxillofac Surg. 2014;40(6):272-277.
8. Adeel M, Rajput MSA, Arain AA, Baloch M, Khan M. Ameloblastoma: Management and outcome. Cureus. 2018; 10:e3437.
9. Batstone MD. Reconstruction of major defects of the jaws. Aust Dent J. 2018;63(S1):S108-S113.
10. Torres Y, Raoul G, Lauwers L, Ferri J. The use of onlay bone grafting for implant restoration in the extremely atrophic anterior maxilla: A case series. 3D Print Med. 2020;6:1.
11. Hung CL, Yang JC, Chang WJ, Hu CY, Lin YH, Huang CH, et al. In vivo graft performance of an improved bone substitute composed of poor crystalline hydroxyapatite based biphasic calcium phosphate. Dent Mater J. 2011;30(1):21-28.
12. Wakimoto M, Ueno T, Hirata A, Iida S, Aghaloo T, Moy PK. Histologic evaluation of human alveolar sockets treated with an artificial bone substitute material. J Craniofac Surg. 2011:22(2):490-493.
13. Miles KB, Maerz T, Matthew HWT. Scalable MSC-derived bone tissue modules: in vitro assessment of differentiation, matrix deposition, and compressive load bearing. Acta Biomater. 2019;95:395-407.
14. Lee YC, Chan YH, Hsieh SC, Lew WZ, Feng SW. Comparing the osteogenic potentials and bone regeneration capacities of bone marrow and dental pulp mesenchymal stem cells in a rabbit calvarial bone defect model. Int J Mol Sci. 2019;20(20):50165.
15. Osugi M, Katagiri W, Yoshimi R, Inukai T, Hibi H, Ueda M. Conditioned media from mesenchymal stem cells enhanced bone regeneration in rat calvarial bone defects. Tissue Eng Part A. 2012;18(13-14):1479-1489.
16. Kaigler D, Pagni G, Park CH, Braun TM, Holman LA, Yi E, et al. Stem cell therapy for craniofacial bone regeneration: A randomized, controlled feasibility trial. Cell Transplant. 2013;22(5):767-777.
17. Gjerde C, Mustafa K, Hellem S, Rojewski M, Gjengedal H, Yassin MA, et al. Cell therapy induced regeneration of severely atrophied mandibular bone in a clinical trial. Stem Cell Res Ther. 2018;9(1):213.
18. Shimizu S, Tsuchiya S, Hirakawa A, Kato K, Ando M, Mizuno M, et al. Design of a randomized controlled clinical study of tissue-engineered osteogenic materials using bone marrow-derived mesenchymal cells for maxillomandibular bone defects in Japan: The TEOM study protocol. BMC Oral Health. 2019;19(1):69.
19. Langer R, Vacanti JP. Advances in tissue engineering. J Pediatr Surg. 2016;51(1):8-12.
20. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816-821.
21. Cho SW, Kim S, Kim JM, Kim JS. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol. 2013;31(3):230-232.
22. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339(6121):819-823.
23. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339(6121):823-826.
24. Yu SJ, Kim HJ, Lee ES, Park CG, Cho SJ, Jeon SH. β-Catenin Accumulation is associated with increased expression of nanog protein and predicts maintenance of msc self-renewal. Cell Transplant. 2017: 26(2):365-377.
25. Pittenger MF, Discher DE, Peault BM, Phinney DG, Hare JM, Caplan AI. Mesenchymal stem cell perspective: Cell biology to clinical progress. NPJ Regen Med. 2019;4:22.
26. Zomer HD, Vidane AS, Gonçalves NN, Ambrósio CE. Mesenchymal and induced pluripotent stem cells: General insights and clinical perspectives. Stem Cells Cloning. 2015;8:125-134.
27. Neri S. Genetic stability of mesenchymal stromal cells for regenerative medicine applications: A fundamental biosafety aspect. Int J Mol Sci. 2019;20(10):2406.
28. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284(5411):143-147.
29. Zhang W, Dong R, Diao S, Du J, Fan Z, Wang F. Differential long noncoding RNA/mRNA expression profiling and functional network analysis during osteogenic differentiation of human bone marrow mesenchymal stem cells. Stem Cell Res Ther. 2017;8(1):30.
30. Lin HR, Heish CW, Liu CH, Muduli S, Li HF, Higuchi A, et al. Purification and differentiation of human adipose-derived stem cells by membrane filtration and membrane migration methods. Sci Rep. 2017;7:40069.
31. Pettersson LF, Kingham PJ, Wiberg M, Kelk P. in vitro osteogenic differentiation of human mesenchymal stem cells from jawbone compared with dental tissue. Tissue Eng Regen Med. 2017; 14(6):763-774.
32. Bieback K, Kern S, Klüter H, Eichler H. Critical parameters for the isolation of mesenchymal stem cells from umbilical cord blood. Stem Cells. 2004;22(4):625-634.
33. Guilak F, Pferdehirt L, Ross AK, Choi YR, Collins K, Nims RJ, et al. Designer stem cells: Genome engineering and the next generation of cell-based therapies. J Orthop Res. 2019;37(6):1287-1293.
34. Jiang W, Lian W, Chen J, Li W, Huang J, Lai B, et al. Rapid identification of genome-edited mesenchymal stem cell colonies via Cas9. Biotechniques. 2019;66(5):231-234.
35. Shahabipour F, Oskuee RK, Shokrgozar MA, Naderi-Meshkin H, Goshayeshi L, Bonakdar S. CRISPR/Cas9 mediated GFP-human dentin matrix protein 1 (DMP1) promoter knock-in at the ROSA26 locus in mesenchymal stem cell for monitoring osteoblast differentiation. J Gene Med. 2020; 22(12):e3288.
36. Narai T, Watase R, Nakayama Y, Kodani I, Inoue T, Kokura K. Establishment of human immortalized mesenchymal stem cells lines for the monitoring and analysis of osteogenic differentiation in living cells. Heliyon. 2020;6(10):e05398.
37. Okamoto T, Aoyama T, Nakayama T, Nakamata T, Hosaka T, Nishijo K, et al. Clonal heterogeneity in differentiation potential of immortalized human mesenchymal stem cells. Biochem Biophys Res Commun. 2002;295:354-361.
38. Fujita S, Toguchida J, Morita Y, Iwata H. Clonal analysis of hematopoiesis-supporting activity of human mesenchymal stem cells in association with Jagged 1 expression and osteogenic potential. Cell Transplant. 2008;17(10-11):1169-1179.
39. Kim SJ, Shin YW, Yang KH, Kim SB, Yoo MJ, Han SK, et al. A multi-center, randomized, clinical study to compare the effect and safety of autologous cultured osteoblast (Ossron) injection to treat fractures. BMC Musculoskelet Disord. 2009;10:20.