Ex vivo expansion of immature 4-hydroperoxycyclophosphamide-resistant progenitor cells from G-CSF-mobilized peripheral blood

AbstractThe application of ex vivo expansion to cell products pharmacologically purged in vitro may provide sufficient numbers of cells for rapid engraftment in a product with reduced tumor burden. To pursue this possibility we evaluated the effect of 4-hydroperoxycyclophosphamide (4-HC) treatment on granulocyte colony-stimulating factor-mobilized peripheral blood stem cells (G-PBSC) and their subsequent expansion potential. A small number of G-PBSC CD34+ cells are resistant to 4-HC and are phenotypically and functionally immature. 4-HC-resistant G-PBSC cells are CD34+ bright, CD38+/-, DR(lo), CD13(lo), CD33-, CD71-, and rhodamine dull. In six experiments, treating G-PBSC with 60 microg/mL of 4-HC at 37 degrees C for 30 minutes reduced the number of colony-forming units (CFUs) per 5000 CD34+ cells by 96.3% (from 1333 +/- 137 to 46.5 +/- 11). This purging also reduced the frequency of 5-week long-term culture initiating cells (LTC-ICs) from 1/39 (range 1/27 to 1/62) to <1/1680 (range 1/1180 to 1/2420). Ex vivo expansion cultures were used to compare the proliferative potential of treated and untreated CD34+ cells. These cells were cultured with either the HS-5 stromal cell line serum-deprived conditioned media supplemented with 10 ng/mL kit ligand (HS-5CM/KL) or a recombinant growth factor mix (GFmix) containing 10 ng/mL each of interleukin (IL)-1, IL-3, IL-6, KL, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and 3 U/mL of erythropoietin. Culturing untreated CD34+ G-PBSC with 10% HS-5CM/KL increased total nucleated cells by 460-fold after 15 days. Progenitors, which were measured as CFUs, also increased by 47-fold over the same period. More significantly, culturing the 4-HC-treated CD34+ cells with HS-5/KL increased CFUs 98-fold and the nucleated cells increased 4573-fold. The absolute number of CFUs present after expansion of the 4-HC-resistant cells with HS-5CM/KL was threefold higher than that detected before purging and significantly higher than that obtained with GFmix. These data indicate that G-PBSC contain a very immature pool of cells not detectable using the 5-week LTC-IC assay, but have extremely high proliferative potential. Additionally, pharmacological purging of G-PBSC greatly reduces mature cells while retaining an immature population. Also significant is the finding that supernatant from the HS-5 bone marrow stromal cell line plus KL can fully regenerate progenitors from the 4-HC-resistant CD34+ G-PBSC.Biol Blood Marrow Transplant 1998;4(2):61-8

Characteristics of Stem Cells Derived from the Degenerated Human Intervertebral Disc Cartilage Endplate

Mesenchymal stem cells (MSCs) derived from adult tissues are an important candidate for cell-based therapies and regenerative medicine due to their multipotential differentiation capability. MSCs have been identified in many adult tissues but have not reported in the human intervertebral disc cartilage endplate (CEP). The initial purpose of this study was to determine whether MSCs exist in the degenerated human CEP. Next, the morphology, proliferation capacity, cell cycle, cell surface epitope profile and differentiation capacity of these CEP-derived stem cells (CESCs) were compared with bone-marrow MSCs (BM-MSCs). Lastly, whether CESCs are a suitable candidate for BM-MSCs was evaluated. Isolated cells from degenerated human CEP were seeded in an agarose suspension culture system to screen the proliferative cell clusters. Cell clusters were chosen and expanded in vitro and were compared with BM-MSCs derived from the same patient. The morphology, proliferation rate, cell cycle, immunophenotype and stem cell gene expression of the CESCs were similar to BM-MSCs. In addition, the CESCs could be induced into osteoblasts, adipocytes, chondrocytes, and are superior to BM-MSCs in terms of osteogenesis and chondrogenesis. This study is first to demonstrate the presence of stem cells in the human degenerated CEP. These results may improve our understanding of intervertebral disc (IVD) pathophysiology and the degeneration process, and could provide cell candidates for cell-based regenerative medicine and tissue engineering