Background: Bone marrow aspirated from the iliac crest contains progenitor cells that can be used to obtain bone-healing of nonunions. However, there is little available information regarding the number and concentration of these cells that are necessary to obtain bone repair. The purpose of this study was to evaluate the number and concentration of progenitor cells that were transplanted for the treatment of nonunion, the callus volume obtained after the transplantation, and the clinical healing rate. Methods: Marrow was aspirated from both anterior iliac crests, concentrated on a cell separator, and then injected into sixty noninfected atrophic nonunions of the tibia. Each nonunion received a relatively constant volume of 20 cm3 of concentrated bone marrow. The number of progenitor cells that was transplanted was estimated by counting the fibroblast colony-forming units. The volume of mineralized bone formation was determined by comparing preoperative computerized tomography scans with scans performed four months following the injection. Results: The aspirates contained an average (and standard deviation) of 612 ± 134 progenitors/cm3 (range, 12 to 1224 progenitors/cm3) before concentration and an average of 2579 ± 1121 progenitors/cm3 (range, 60 to 6120 progenitors/cm3) after concentration. An average total of 51 × 103 fibroblast colony-forming units was injected into each nonunion. Bone union was obtained in fifty-three patients, and the bone marrow that had been injected into the nonunions of those patients contained >1500 progenitors/cm3 and an average total of 54,962 ± 17,431 progenitors. The concentration (634 ± 187 progenitors/cm3) and the total number (19,324 ± 6843) of progenitors injected into the nonunion sites of the seven patients in whom bone union was not obtained were both significantly lower (p = 0.001 and p < 0.01, respectively) than those in the patients who obtained bone union. The volume of the mineralized callus measured at four months on the computerized tomography scans of the patients who had union ranged from 0.8 to 5.3 cm3 (mean, 3.1 cm3). There was a positive correlation between the volume of mineralized callus at four months and the number (p = 0.04) and concentration (p = 0.01) of fibroblast colony-forming units in the graft. There was a negative correlation between the time needed to obtain union and the concentration of fibroblast colony-forming units in the graft (p = 0.04). Conclusions: Percutaneous autologous bone-marrow grafting is an effective and safe method for the treatment of an atrophic tibial diaphyseal nonunion. However, its efficacy appears to be related to the number of progenitors in the graft, and the number of progenitors available in bone marrow aspirated from the iliac crest appears to be less than optimal in the absence of concentration. Level of Evidence: Therapeutic Level III. See Instructions to Authors for a complete description of levels of evidence.

Clinical-grade human mesenchymal stromal cells (MSCs) have been expanded in vitro for tissue engineering or immunoregulatory purposes without standardized culture conditions or release criteria. Although human MSCs show poor susceptibility for oncogenic transformation, 2 recent studies described their capacity to accumulate chromosomal instability and to give rise to carcinoma in immunocompromised mice after long-term culture. We thus investigated the immunologic and genetic features of MSCs expanded with fetal calf serum and fibroblast growth factor or with platelet lysate in 4 cell-therapy facilities during 2 multicenter clinical trials. Cultured MSCs showed a moderate expression of human leukocyte antigen-DR without alteration of their low immunogenicity or their immunomodulatory capacity. Moreover, some transient and donor-dependent recurring aneuploidy was detected in vitro, independently of the culture process. However, MSCs with or without chromosomal alterations showed progressive growth arrest and entered senescence without evidence of transformation either in vitro or in vivo.

In vitro RBC production from stem cells could represent an alternative to classic transfusion products. Until now the clinical feasibility of this concept has not been demonstrated. We addressed the question of the capacity of cultured RBCs (cRBCs) to survive in humans. By using a culture protocol permitting erythroid differentiation from peripheral CD34+ HSC, we generated a homogeneous population of cRBC functional in terms of their deformability, enzyme content, capacity of their hemoglobin to fix/release oxygen, and expression of blood group antigens. We then demonstrated in the nonobese diabetes/severe combined immunodeficiency mouse that cRBC encountered in vivo the conditions necessary for their complete maturation. These data provided the rationale for injecting into one human a homogeneous sample of 1010 cRBCs generated under good manufacturing practice conditions and labeled with 51Cr. The level of these cells in the circulation 26 days after injection was between 41% and 63%, which compares favorably with the reported half-life of 28 ± 2 days for native RBCs. Their survival in vivo testifies globally to their quality and functionality. These data establish the proof of principle for transfusion of in vitro–generated RBCs and path the way toward new developments in transfusion medicine. This study is registered at http://www.clinicaltrials.gov as NCT0929266.