Cytotherapy, 2014; 16: 454e459

Presence of osteoclast precursor cells during ex vivo expansion of bone marrow-derived mesenchymal stem cells for autologous use in cell therapy  J. MINGUELL CAROLINA ALLERS, GABRIEL P. LASALA & JOSE TCA Cellular Therapy, Covington, Louisiana, USA Abstract Background aims. To obtain a cell product competent for clinical use in terms of cell dose and biologic properties, bone marrow-derived mesenchymal stem cells (MSCs) must be expanded ex vivo. Methods. A retrospective analysis was performed of records of 76 autologous MSC products used in phase I or II clinical studies performed in a cohort of cardiovascular patients. In all cases, native MSCs present in patient bone marrow aspirates were separated and expanded ex vivo. Results. The cell products were classified in two groups (A and B), according to biologic properties and expansion time (ex vivo passages) to reach the protocol-established cell dose. In group A, the population of adherent cells obtained during the expansion period (2  1 passages) was composed entirely of MSCs and met the requirements of cell number and biologic features as established in the respective clinical protocol. In group B, in addition to MSCs, we observed during expansion a high proportion of ancillary cells, characterized as osteoclast precursor cells. In this case, although the biologic properties of the resulting MSC product were not affected, the yield of MSCs was significantly lower. The expansion cycles had to be increased (3  1 passages). Conclusions. These results suggest that the presence of osteoclast precursor cells in bone marrow aspirates may impose a limit for the proper clinical use of ex vivo expanded autologous bone marrow-derived MSCs. Key Words: cell therapy, ex vivo expansion, mesenchymal stem cells, osteoclast precursor

Introduction During the last decade, mesenchymal stem cells (MSCs) have become a very challenging tool for use in clinical studies. Several attributes of MSCs make their use attractive, including the ability to self-renew, differentiate into several cell lineages, produce growth factors and extracellular matrix molecules and modulate the immune response (1e4). Adult bone marrow is the most recurrent source of MSCs for use in clinical studies. Because the frequency of MSCs in a bone marrow aspirate (“native MSC”) is extremely low, (1 MSC for each 10,000e100,000 mononuclear cells (5)), it has been accepted that for suitable clinical use the load of native cells in the bone marrow must be increased ex vivo. So far, the most widespread method for expansion, particularly in the autologous setting, is based on the capacity of MSCs to attach to plastic surfaces and, after exposure to nutrients, initiate a process of cell division, mainly regulated by cell-tocell contacts. As soon as the number of dividing cells in the culture is high enough (confluence), cells enter a quiescence condition; at this point, MSCs can be

gently dislodged from the culture vessel and then set again in culture, and the expansion procedure (passages) is re-initiated until the required number of cells is attained. Several reports have recognized that the functional feasibility of a MSC product obtained after ex vivo expansion may be limited by several factors, including number of passages during expansion and a patient’s consumption of diverse prescription medications (4,6). To evaluate further the abovementioned limitations accompanying ex vivo expansion of MSCs, we performed a retrospective analysis of records dealing with the functional feasibility (cell number and biologic features) of autologous bone marrow-derived MSC products prepared from a cohort of 76 patients with cardiovascular disease recruited to participate in phase I or II clinical trials performed at this institution. The results of this analysis put forward the notion that the presence of “contaminant” cells visualized during cell expansion and identified as osteoclast precursor cells (OPCs), may pose a hurdle to generate a final MSC product containing the reliable cell number as defined by the

Correspondence: Carolina Allers, PhD, 101 Judge Tanner Boulevard, Suite 502, Covington, LA 70433, USA. E-mail: [email protected] (Received 19 March 2013; accepted 10 August 2013) ISSN 1465-3249 Copyright Ó 2014, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Presence of osteoclast precursor cells during expansion of MSCs respective clinical protocol. However, the presence of the contaminant cells does not affect the biologic features of the resulting population of ex vivo expanded bone marrow-derived MSCs. Methods Bone marrow aspiration Bone marrow was aspirated from the iliac crest of 76 patients with cardiovascular disease enrolled in clinical trials NCT00643981, NCT00790764, NCT00548613, NCT00518401 and NCT00721006 ( In each case, 71  27 mL was aspirated and sent to a Good Manufacturing Practices facility for preparation of ex vivo expanded MSCs. Ex vivo expansion of bone marrow “native” MSCs Autologous bone marrow-derived MSCs were prepared and processed as previously described (7), following a precise and restrictive methodology described in the U.S. Food and Drug Administration-approved clinical study to obtain, in the shortest time, a population of MSCs ready to be infused immediately to the patient. Briefly, the fraction of bone marrow-derived mononuclear cells was suspended in culture medium containing a-minimum essential medium (Invitrogen, Carlsbad, CA, USA) containing 20% fetal bovine serum (HyClone, Logan, UT, USA). The cell suspension was seeded (1  106 cells/cm2) and incubated (5% CO2 “personal” cell culture incubator). At 3 days after initiation of the cell expansion, a few MSCs were observed attached to the culture vessel, whereas most were non-adherent floating cells, which were progressively eliminated after each medium change (every 3 days). On reaching approximately 80% confluence and to prevent contact inhibition and spontaneous differentiation (8), attached MSCs were enzymatically dislodged (0.25% trypsin solution; HyClone), re-suspended in culture medium, seeded (10  103 cells/cm2) and cultured again. The last described sequence of ex vivo expansion (a passage) was repeated until completion of the required cell number for patient infusion (0.1  106e0.4  106 MSC/kg body weight), as described in the respective clinical protocol. Cell characterization According to the respective clinical protocol, MSCs visualized under an inverted light microscope exhibited a fibroblast-like morphology of fully adherent cells. The immunophenotype was evaluated


by flow cytometry after immunolabeling with antibodies CD34 (BD Biosciences, San Jose, CA, USA), CD45 (BD Biosciences), CD73 (BD Pharmingen, San Jose, CA, USA), CD90 (BD Pharmingen) and CD105 (AbD Serotec, Oxford, UK) (7,9). OPCs were characterized by visualization under an inverted light microscope as a group of attached round or oval cells, with one or more nuclei and smooth borders. These cells were characterized by high expression of tartrate-resistant acid phosphatase (TRAP; Sigma-Aldrich, St Louis, MO, USA) and an immunophenotype showing expression of antigen CD45 (BD Biosciences) but not of CD51/61 (BioLegend, San Diego, CA, USA) and CD73 (BD Pharmingen). Analysis was performed using a LSRII analytic flow cytometer (Tulane Primate Research Center, Covington, LA, USA) after cell immunolabeling with commercial antibodies (BD Biosciences, Biolegend and BD Pharmingen).

Removal of OPCs from MSC cultures during ex vivo expansion To remove or minimize the content of OPCs in MSC cultures during ex vivo expansion, we took advantage of a recurring microscopic observation that although trypsin-dependent detachment of MSCs from a culture vessel occurs rapidly (3e5 min), OPCs require a longer exposure period to trypsin (10e15 min). When OPCs were visualized during manufacture of the cell product, MSC detachment with trypsin was performed for

Presence of osteoclast precursor cells during ex vivo expansion of bone marrow-derived mesenchymal stem cells for autologous use in cell therapy.

To obtain a cell product competent for clinical use in terms of cell dose and biologic properties, bone marrow-derived mesenchymal stem cells (MSCs) m...
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