Primary Lin^neg/CD45^neg/CD271^pos/PDGFRα^low/Neg Stroma Stem Cells In Adult Human Bone Marrow Show a Distinct Molecular Signature and Potent Hematopoiesis-Supporting Function

Human bone marrow contains a rare population of mesenchymal stroma stem cells (BM-MSC) that are capable of generating all skeletal lineages such as osteoblasts, adipocytes, chondrocytes and fibroblastic reticular cells. In vivo, BM-MSC are essential constituents of the hematopoietic stem cell niche, thus playing an important role in supporting, maintaining and controlling hematopoiesis. Despite their central role in bone marrow physiology, little is known about the primary MSC. This is mainly due to the fact that a precise phenotypical definition of BM-MSC has been lacking so far. We were first to report that low/negative expression of PDGFRα on lin^neg/CD45^neg/CD271^pos cells allowed identify the candidate primary stromal stem cell population in adult human bone marrow, i.e lin^neg/CD45^neg/CD271^pos/PDGFRα^low/neg cells were highly enriched in CFU-F (1 in 4) and showed all typical BM-MSC properties in-vitro and in-vivo (Li et al. Blood, 2012, 120:3460).

This key finding now facilitated to characterize the molecular signature and hematopoiesis-supporting function of primary BM-MSC. Illumina Human HT-12 expression v4 BeadChips containing 48,107 probes were utilized to perform gene expression profiling analysis comparing lin^neg/CD45^neg/CD271^pos/PDGFRα^low/neg and lin^neg/CD45^neg/CD271^pos/PDGFRα^pos cells (five independent samples in each group). The expression of 365 genes, including 22 novel surface antigens, such as ITGB5, ANTXR2, CD63, CD230, APLNR and CD74 was significantly higher in the PDGFRα^low/neg subset, whereas 65 genes showed reduced expression. The expression of cell cycle inhibitor genes, such as cyclin-dependent kinase inhibitor 1 (CDKN1A), B cell translocation gene family, member 3 (BTG3) and dual specificity protein phosphatase 3 (DUSP3), was increased in the PDGFRα^low/neg population, indicating a quiescent status of the majority of the cells, which was confirmed by cell cycle analysis (Ki67/DAPI staining; 96.8 ± 2.9% in G0 phase, n=3). In accordance with the hematopoiesis maintenance function, expression of genes encoding ECM proteins, such as laminin subunit alpha-4 (LAMA4), adrenomedullin (ADM) and collagen type I alpha 1 (COL1A1) was also significantly higher in PDGFRα^low/neg cells. Additionally, quantitative RT-PCR analysis revealed that PDGFRα^low/neg cells expressed high levels of CXCL12, VCAM1 and osteopontin, i.e. genes known to be related to hematopoietic stem cell (HSC) supportive function.

In order to test the hematopoiesis-supporting capacity of primary BM-MSC, 7-day co-culture experiments with human cord blood CD34⁺ cells were performed in SCF, TPO, and FL-supplemented serum-free medium. CD34⁺ cells were evenly distributed and mainly found in close contact with stromal cells when cocultured with lin^neg/CD45^neg/CD271^pos/PDGFRα^low/neg cells, compared to cultures with lin^neg/CD45^neg/CD271^neg/PDGFRα^neg cells and lin^neg/CD45^neg/CD271^pos/PDGFRα^pos cells. Co-culture with PDGFRα^low/neg cells did not only amplify total hematopoietic cells (38.8 ± 14.4 fold) but, importantly, also expanded CD34⁺ cells very effectively (10.2 ± 3.8 fold, compared to 2.4 ± 0.2, 3.6 ± 0.7, and 3.2 ± 0.8 fold for no stroma, CD271^neg/PDGFRα^neg, and PDGFRα^pos cells, respectively; p<0.05, n=3). Moreover, the percentage of CD34⁺ cells was 2-3 fold higher in PDGFRα^low/neg co-cultures. Xenotransplantation experiments investigating the expansion of transplantable hematopoietic stem cells are currently under way.

Taken together, primary lin^neg/CD45^neg/CD271^pos/PDGFRα^low/neg stroma stem cells show a distinct molecular signature and potent hematopoiesis-supporting function. The study of primary BM-MSC will lead to a better understanding of the nature and the physiological role of these cells in the human bone marrow in-situ.