Analysis of the Karyotype of Expanded Human Adipose-Derived Stem Cells for Bone Reconstruction of the Maxillo-Facial Region

Abstract

Mesenchymal stem cells (MSC) and adipose-derived stem cells (ASC) were recently proposed for bone maxillofacial reconstruction in association with biomaterials. For this application MSC must be ex-vivo expanded in order to obtain, for a given volume of implanted biomaterial, a relevant number of bone forming cells. Previously conducted pre-clinical studies suggested that a concentration of 6 x 10 8 ASC associated with 900 mg of anorganic bovine bone (ABB) could be effective for human maxillary sinus floor elevation. A keystone issue to guarantee the quality and safety of Advanced Therapy Medicinal Products containing expanded MSC and ASC is their chromosome stability in culture: this topic has been widely investigated and conflicting results have been published. Abnormal karyotype of human ex-vivo expanded MSC and ASC was found by some authors, while, at the same time, several other studies showed the MSC and ASC karyotype to be normal. It is therefore important that all the results obtained on MSC andASC karyotype analysis be published. Given this context, the aim of this manuscript, aim of this manuscript is to verify the karyotype stability of ASC in view oftheir applications in clinical trials. ASC obtained from the adipose tissue of 4 donors were expanded over extended culture time. Based on previous ASC expansions we hypothesized to be able to obtain 6 x lOS cells by passage 7. Karyotype analysis 000 metaphases was planned to be investigated at passage 2, 7, and 15 in all the cultures. No abnormalities were found in the karyotype of two donors at all the passages tested, while a translocation was found in 2 metaphases of a donor at passage 7, but not at passage 15, and in the fourth donor in 5 metaphases a trisomy was found at passage 15. Chromosomal abnormalities were detected only after extended ASC expansion. Whether these anomalies can be related to risk for the patient's safety will have to be demonstrated by in-vivo studies.

Variation in Osteogenic Differentiation Capacities of Adiposederived Stromal Cells by Anatomic Depot

“…For one, the literature has shown that excessive adiposity has been associated with increased incidence of breast cancer in postmenopausal women, likely due to IL-6 secretion by ASC which is hypothesized to promote migration and invasion of breast cancer cells [87,88] . Another study performed ex vivo detected chromosomal abnormalities after extended ASC expansion in culture [89] , similar to the findings of other groups that culture propagation of MSC introduces genetic instability and tumorigenicity [90] . Furthermore, one ASC study pertaining to bone formation in canine by Kang et al found cytologic abnormalities along with increased proliferation associated with ASC [91,92] .…”

Derivation of Canine Induced Pluripotent Stem Cells

“…; Bellotti et al. ). To our knowledge, karyotype stability has not been studied extensively in dog AdMS cells, but published studies to date suggest that, like human AdMS cells, dog cells have a stable karyotype (Vieira et al.…”

“…The AdMS cells used to generate the canine iPSCs had an abnormal karyotype, displaying both aneuploidy and the appearance of metacentric chromosomes at low passage (P5). Human AdMS cells have been shown to exhibit karyotype abnormalities but only following prolonged culture periods (Izadpanah et al 2008;Bellotti et al 2013). To our knowledge, karyotype stability has not been studied extensively in dog AdMS cells, but published studies to date suggest that, like human AdMS cells, dog cells have a stable karyotype (Vieira et al 2010).…”

Adipose-Derived Stem Cells: A Review of Signaling Networks Governing Cell Fate and Regenerative Potential in the Context of Craniofacial and Long Bone Skeletal Repair

“…Other obstacles that must be addressed include the need for development of suitable serum free media for these cells as fetal bovine serum (FBS) is not recommended for clinical therapies given the risk of contamination and infection. Current strategies require ex vivo expansion of cells for two or three weeks rendering them susceptible to the possibility of genomic instability in culture [98]. Attempts at designing devices that enable a single step harvest, manipulation and grafting are therefore needed and would obviate the need for cell culture and the attendant risks of using FBS [99,100].…”