Pathological Heterotopic Ossifications in Brain or Spinal Cord Injured Patients: From Ectopic Bone to Hematopoietic Stem Cell Niche Development

Heterotopic ossification (HO) is a pathology characterized by the formation of ectopic bone in soft tissues generally in muscles surrounding joints such hip, knee, elbow or shoulder. The etiology of this pathology is not known but it is well established that the risk of HO is increased after traumatic brain or spinal cord injuries and also after fractures or burns. Patients with HOs suffer from pain and the range of motion from limbs with ectopic bone is highly reduced. Currently, the only effective treatment for HOs is surgery but recurrences are frequent underpinning that a better understanding of its pathophysiology is required to cure patients.

Hematopoietic marrow-like tissue is frequently observed on histological section of HO. Therefore, we investigated whether HOs from patients with traumatic brain or spinal cord injury represent an ectopic model of hematopoietic stem cell (HSC) niche as defined by the presence of HSC-supportive stromal cells.

HOs were obtained from patients after surgical resection in Garches Hospital (France) with their informed consent. HOs were cut in small pieces and hematopoietic as well as endothelial cells were extracted from the HO marrow by gentle washing. Mononuclear cells were then recovered by ficoll gradient and were either directly analyzed by flow cytometry or purified by immunomagnetic selection for in vitro and in vivo experiments. Mesenchymal stromal cells (MSCs) were obtained by plastic adherence from HO explants.

We first confirmed the presence of hematopoietic cells from different lineages in HO marrow such as neutrophils, monocytes/macrophages, B and T lymphocytes as evidenced by membrane expression of CD45, CD15, CD11/CD14, CD19, CD3/CD4 and CD3/CD8 antigens. HSC presence within HO marrow was further demonstrated by: (i) in vitro clonogenic assays, (ii) presence of quiescent CD45⁺CD34⁺CD38^- cells with a side-population phenotype and, (iii) in vivo human hematopoietic reconstitution assays in immunodeficient NSG mice.

We also show that human HO marrow contained functional MSCs and endothelial progenitors (EPs) as demonstrated by phenotypic and functional analyses. MSC expressing the classical mesenchymal markers CD73, CD90, CD105 and capable to in vitro and in vivo differentiate into osteoblasts, adipocytes and chondrocytes could be isolated from human HOs. These MSCs were also able to support in vitro long term human hematopoiesis and in vivo murine hematopoiesis when seeded on hydroxyapatite scaffolds and implanted into nude mice. Endothelial progenitors with a characteristic CD31⁺CD144⁺CD34⁺CD45^-CD90⁺ phenotype were detected in the mononuclear cell fraction from HO marrows. After sorting, these cells were able to form colonies on plastic and vascular networks when cultured in a Matrigel and to express high levels of VCAM-1 and ICAM-1 adhesion molecules after TNFα stimulation, confirming their EP functional capability.

In conclusion, we show for the first time that human HOs contain a marrow tissue with quiescent HSC that can proliferate and differentiate within a suitable and functional mesenchymal and endothelial microenvironment, acknowledging that HO marrows are ectopic HSC niches. We have recently demonstrated the involvement of macrophages in the development of neurogenic HOs in spinalized mice¹. Taking into account the role of immune cells and the neurogenic origin of human HOs, our results bring new evidences for the role of interactions between bone and neuro-immune systems in the initiation and development of adult hematopoiesis. Mechanisms underlying these processes are currently studied in our laboratory.

¹ Genet F et al., J Pathol. 2015 Jun;236(2):229-40