Scalable Manufacturing of Human Hematopoietic Stem/Progenitor Cells Exploiting a Co-culture Platform with Mesenchymal Stromal Cells.

In the context of hematopoietic cell transplantation, hematopoietic stem/progenitor cells (HSPC) from the umbilical cord blood (UCB) present several advantages compared to adult sources including higher proliferative capacity, abundant availability and ease of collection, non-risk and painless harvesting procedure, and lower risk of graft-versus-host disease. However, the therapeutic utility of UCB HSPC has been limited to pediatric patients due to the low cell frequency per unit of UCB. The development of efficient and cost-effective strategies to generate large numbers of functional UCB HSPC ex vivo would boost all current and future medical uses of these cells. Herein, we describe a scalable serum-free co-culture system for the expansion of UCB-derived CD34+-enriched cells using microcarrier-immobilized human bone marrow-derived mesenchymal stromal cells as feeder cells.

Developing a co-culture system for effective megakaryo/thrombopoiesis from umbilical cord blood hematopoietic stem/progenitor cells.

BACKGROUND AIMS: Platelet transfusion can be a life-saving procedure in different medical settings. Thus, there is an increasing demand for platelets, of which shelf-life is only 5 days. The efficient ex vivo biomanufacturing of platelets would allow overcoming the shortages of donated platelets. METHODS: We exploited a two-stage culture protocol aiming to study the effect of different parameters on the megakaryo/thrombopoiesis ex vivo. In the expansion stage, human umbilical cord blood (UCB)-derived CD34(+)-enriched cells were expanded in co-culture with human bone marrow mesenchymal stromal cells (BM-MSCs). The megakaryocytic commitment and platelet generation were studied, considering the impact of exogenous addition of thrombopoietin (TPO) in the expansion stage and a cytokine cocktail (Cyt) including TPO and interleukin-3 in the differentiation stage, with the use of different culture medium formulations, and in the presence/absence of BM-MSCs (direct versus non-direct cell-cell contact). RESULTS: Our results suggest that an early megakaryocytic commitment, driven by TPO addition during the expansion stage, further enhanced megakaryopoiesis. Importantly, the results suggest that co-culture with BM-MSCs under serum-free conditions combined with Cyt addition, in the differentiation stage, significantly improved the efficiency yield of megakaryo/thrombopoiesis as well as increasing %CD41, %CD42b and polyploid content; in particular, direct contact of expanded cells with BM-MSCs, in the differentiation stage, enhanced the efficiency yield of megakaryo/thrombopoiesis, despite inhibiting their maturation. CONCLUSIONS: The present study established an in vitro model for the hematopoietic niche that combines different biological factors, namely, the presence of stromal/accessory cells and biochemical cues, which mimics the BM niche and enhances an efficient megakaryo/thrombopoiesis process ex vivo.

Stem cell bioengineering strategies to widen the therapeutic applications of haematopoietic stem/progenitor cells from umbilical cord blood.

Umbilical cord blood (UCB) transplantation has observed a significant increase in recent years, due to the unique features of UCB haematopoietic stem/progenitor cells (HSCs) for the treatment of blood-related disorders. However, the low cell numbers available per UCB unit significantly impairs the widespread use of this source for transplantation of adult patients, resulting in graft failure, delayed engraftment and delayed immune reconstitution. In order to overcome this issue, distinct approaches are now being considered in clinical trials, such as double-UCB transplantation, intrabone injection or ex vivo expansion. In this article the authors review the current state of the art, future trends and challenges on the ex vivo expansion of UCB HSCs, focusing on culture parameters affecting the yield and quality of the expanded HSC grafts: novel HSC selection schemes prior to cell culture, cytokine/growth factor cocktails, the impact of biochemical factors (e.g. O2 ) or the addition of supportive cells, e.g. mesenchymal stem/stromal cell (MSC)-based feeder layers) were addressed. Importantly, a critical challenge in cellular therapy is still the scalability, reproducibility and control of the expansion process, in order to meet the clinical requirements for therapeutic applications. Efficient design of bioreactor systems and operation modes are now the focus of many bioengineers, integrating the increasing 'know-how' on HSC biology and physiology, while complying with the GMP standards for the production of cellular products, i.e. through the use of commercially available, highly controlled, disposable technologies.

Ex vivo expansion of cord blood haematopoietic stem/progenitor cells under physiological oxygen tensions: clear-cut effects on cell proliferation, differentiation and metabolism.

Physiologically low O(2) tensions are believed to regulate haematopoietic stem cell (HSC) functions in the bone marrow (BM; 0-5%). In turn, placenta and umbilical cord are characterized by slightly higher physiological O(2) tensions (3-10%). We hypothesized that O(2) concentrations within this range may be exploited to augment the ex vivo expansion/maintenance of HSCs from umbilical cord (placental) blood (UCB). The expansion of UCB CD34(+) -enriched cells was studied in co-culture with BM mesenchymal stem/stromal cells (MSCs) under 2%, 5%, 10% and 21% O(2). 2% O(2) resulted in a significantly lower CD34(+) cell expansion (25-fold vs 60-, 64- and 92-fold at day 10 for 5%, 21%, 10% O(2), respectively). In turn, 10% O(2) promoted the highest CD34(+) CD90(+) cell expansion, reaching 22 ± 5.4- vs 5.6 ± 2.4- and 5.7 ± 2.0-fold for 2%, 5% and 21% O(2), respectively, after 14 days. Similar differentiation patterns were observed under different O(2) tensions, being primarily shifted towards the neutrophil lineage. Cell division kinetics revealed a higher proliferative status of cells cultured under 10% and 21% vs 2% O(2). Expectedly, higher specific glucose consumption and lactate production rates were determined at 2% O(2) when compared to higher O(2) concentrations (5-21%). Overall, these results suggest that physiological oxygen tensions, in particular 10% O(2), can maximize the ex vivo expansion of UCB stem/progenitor cells in co-culture with BM MSCs. Importantly, these studies highlight the importance of exploiting knowledge of the intricate microenvironment of the haematopoietic niche towards the definition of efficient and controlled ex vivo culture systems capable of generating large HSCs numbers for clinical applications.

Differentiation of human umbilical cord matrix mesenchymal stem cells into neural-like progenitor cells and maturation into an oligodendroglial-like lineage.

Mesenchymal stem cells (MSCs) are viewed as safe, readily available and promising adult stem cells, which are currently used in several clinical trials. Additionally, their soluble-factor secretion and multi-lineage differentiation capacities place MSCs in the forefront of stem cell types with expected near-future clinical applications. In the present work MSCs were isolated from the umbilical cord matrix (Wharton's jelly) of human umbilical cord samples. The cells were thoroughly characterized and confirmed as bona-fide MSCs, presenting in vitro low generation time, high proliferative and colony-forming unit-fibroblast (CFU-F) capacity, typical MSC immunophenotype and osteogenic, chondrogenic and adipogenic differentiation capacity. The cells were additionally subjected to an oligodendroglial-oriented step-wise differentiation protocol in order to test their neural- and oligodendroglial-like differentiation capacity. The results confirmed the neural-like plasticity of MSCs, and suggested that the cells presented an oligodendroglial-like phenotype throughout the differentiation protocol, in several aspects sharing characteristics common to those of bona-fide oligodendrocyte precursor cells and differentiated oligodendrocytes.

A xenogeneic-free bioreactor system for the clinical-scale expansion of human mesenchymal stem/stromal cells.

The large cell doses (>1 × 10(6) cells/kg) used in clinical trials with mesenchymal stem/stromal cells (MSC) will require an efficient production process. Moreover, monitoring and control of MSC ex-vivo expansion is critical to provide a safe and reliable cell product. Bioprocess engineering approaches, such as bioreactor technology, offer the adequate tools to develop and optimize a cost-effective culture system for the rapid expansion of human MSC for cellular therapy. Herein, a xenogeneic (xeno)-free microcarrier-based culture system was successfully established for bone marrow (BM) MSC and adipose tissue-derived stem/stromal cell (ASC) cultivation using a 1L-scale controlled stirred-tank bioreactor, allowing the production of (1.1 ± 0.1) × 10(8) and (4.5 ± 0.2) × 10(7) cells for BM MSC and ASC, respectively, after 7 days. Additionally, the effect of different percent air saturation values (%Airsat ) and feeding regime on the proliferation and metabolism of BM MSC was evaluated. No significant differences in cell growth and metabolic patterns were observed under 20% and 9%Airsat . Also, the three different feeding regimes studied-(i) 25% daily medium renewal, (ii) 25% medium renewal every 2 days, and (iii) fed-batch addition of concentrated nutrients and growth factors every 2 days-yielded similar cell numbers, and only slight metabolic differences were observed. Moreover, the immunophenotype (positive for CD73, CD90 and CD105 and negative for CD31, CD80 and HLA-DR) and multilineage differentiative potential of expanded cells were not affected upon bioreactor culture. These results demonstrated the feasibility of expanding human MSC from different sources in a clinically relevant expansion configuration in a controlled microcarrier-based stirred culture system under xeno-free conditions. The further optimization of this bioreactor culture system will represent a crucial step towards an efficient GMP-compliant clinical-scale MSC production system.

Proliferation extent of CD34+ cells as a key parameter to maximize megakaryocytic differentiation of umbilical cord blood-derived hematopoietic stem/progenitor cells in a two-stage culture protocol.

Co-infusion of ex-vivo generated megakaryocytic progenitors with hematopoietic stem/progenitor cells (HSC/HPC) may contribute to a faster platelet recovery upon umbilical cord blood (UCB) transplantation. A two stage protocol containing cell expansion and megakaryocyte (Mk) differentiation was established using human UCB CD34+-enriched cells. The expansion stage used a pre-established protocol supported by a human bone marrow mesenchymal stem cells (MSC) feeder layer and the differentiation stage used TPO (100 ng/mL) and IL-3 (10 ng/mL). 18% of culture-derived Mks had higher DNA content (>4 N) and were able to produce platelet-like particles. The proliferation extent of CD34+ cells obtained in the expansion stage (FI-CD34+), rather than expansion duration, determined as a key parameter for efficient megakaryocytic differentiation. A maximum efficiency yield (EY) of 48 ± 7.7 Mks/input CD34+ cells was obtained for a FI-CD34+ of 17 ± 2.5, where a higher FI-CD34+ of 42 ± 13 resulted in a less efficient megakaryocytic differentiation (EY of 22 ± 6.7 and 19 ± 4.6 %CD41).

Bioreactor design for clinical-grade expansion of stem cells.

The many clinical trials currently in progress will likely lead to the widespread use of stem cell-based therapies for an extensive variety of diseases, either in autologous or allogeneic settings. With the current pace of progress, in a few years' time, the field of stem cell-based therapy should be able to respond to the market demand for safe, robust and clinically efficient stem cell-based therapeutics. Due to the limited number of stem cells that can be obtained from a single donor, one of the major challenges on the roadmap for regulatory approval of such medicinal products is the expansion of stem cells using Good Manufacturing Practices (GMP)-compliant culture systems. In fact, manufacturing costs, which include production and quality control procedures, may be the main hurdle for developing cost-effective stem cell therapies. Bioreactors provide a viable alternative to the traditional static culture systems in that bioreactors provide the required scalability, incorporate monitoring and control tools, and possess the operational flexibility to be adapted to the differing requirements imposed by various clinical applications. Bioreactor systems face a number of issues when incorporated into stem cell expansion protocols, both during development at the research level and when bioreactors are used in on-going clinical trials. This review provides an overview of the issues that must be confronted during the development of GMP-compliant bioreactors systems used to support the various clinical applications employing stem cells.

Human mesenchymal stem cells from the umbilical cord matrix: successful isolation and ex vivo expansion using serum-/xeno-free culture media.

Mesenchymal stem cells (MSC) could potentially be applied in therapeutic settings due to their multilineage differentiation ability, immunomodulatory properties, as well as their trophic activity. The umbilical cord matrix (UCM) represents a promising source of MSC for biomedical applications. The number of cells isloated per umbilical cord (UC) unit is limited and ex vivo expansion is imperative in order to reach clinically meaningful cell numbers. The limitations of poorly defined reagents (e.g. fetal bovine serum, which is commonly used as a supplement for human MSC expansion) make the use of serum-/xeno-free conditions mandatory. We demonstrated the feasibility of isolating UCM-MSC by plastic adherence using serum-/xeno-free culture medium following enzymatic digestion of UCs, with a 100% success rate. 2.6 ± 0.21 × 10(5) cells were isolated per UC unit, of which 1.9 ± 0.21 × 10(5) were MSC-like cells expressing CD73, CD90, and CD105. When compared to adult sources (bone marrow-derived MSC and adipose-derived stem/stromal cells), UCM-MSC displayed a similar immunophenotype and similar multilineage differentiation ability, while demonstrating a higher expansion potential (average fold increase of 7.4 for serum-containing culture medium and 11.0 for xeno-free culture medium (P3-P6)). The isolation and expansion of UCM-MSC under defined serum-/xeno-free conditions contributes to safer and more effective MSC cellular products, boosting the usefulness of MSC in cellular therapy and tissue engineering.

Toward a clinical-grade expansion of mesenchymal stem cells from human sources: a microcarrier-based culture system under xeno-free conditions.

The immunomodulatory properties of mesenchymal stem cells (MSCs) make them attractive therapeutic agents for a wide range of diseases. However, the highly demanding cell doses used in MSC clinical trials (up to millions of cells/kg patient) currently require labor intensive methods and incur high reagent costs. Moreover, the use of xenogenic (xeno) serum-containing media represents a risk of contamination and raises safety concerns. Bioreactor systems in combination with novel xeno-free medium formulations represent a viable alternative to reproducibly achieve a safe and reliable MSC doses relevant for cell therapy. The main goal of the present study was to develop a complete xeno-free microcarrier-based culture system for the efficient expansion of human MSC from two different sources, human bone marrow (BM), and adipose tissue. After 14 days of culture in spinner flasks, BM MSC reached a maximum cell density of (2.0±0.2)×105 cells·mL⁻¹ (18±1-fold increase), whereas adipose tissue-derived stem cells expanded to (1.4±0.5)×105 cells·mL⁻¹ (14±7-fold increase). After the expansion, MSC expressed the characteristic markers CD73, CD90, and CD105, whereas negative for CD80 and human leukocyte antigen (HLA)-DR. Expanded cells maintained the ability to differentiate robustly into osteoblast, adipocyte, and chondroblast lineages upon directed differentiation. These results demonstrated the feasibility of expanding human MSC in a scalable microcarrier-based stirred culture system under xeno-free conditions and represent an important step forward for the implementation of a Good Manufacturing Practices-compliant large-scale production system of MSC for cellular therapy.

A novel method for human hematopoietic stem/progenitor cell isolation from umbilical cord blood based on immunoaffinity aqueous two-phase partitioning.

A novel cell separation process based on immunoaffinity aqueous two phase systems is presented to isolate and purify CD34(+) stem/progenitor cells directly from the whole umbilical cord blood (UCB). A system, composed of polyethylene glycol and dextran, was evaluated for the selective recovery of CD34(+) cells from UCB. A monoclonal antibody against the CD34 surface antigen was used for the direct partitioning of CD34(+) cells in UCB to the PEG-rich phase. The initial population of CD34(+) cells (0.2% of the initial sample) was enriched to values up to 42% in a single partitioning step, while the majority of contaminant cells were partitioned to the dextran-rich phase (1.37 × 10(-2) < K(P) < 2.76 × 10(-2)). This novel selection method allowed a recovery yield of 95% of CD34(+) cells with a purification factor of 245 and is expected to pave a new way to purify hematopoietic stem/progenitor cells for use in a variety of clinical settings.

Initial CD34+ cell-enrichment of cord blood determines hematopoietic stem/progenitor cell yield upon ex vivo expansion.

Since umbilical cord blood (UCB), contains a limited hematopoietic stem/progenitor cells (HSC) number, successful expansion protocols are needed to overcome the hurdles associated with inadequate numbers of HSC collected for transplantation. UCB cultures were performed using a human stromal-based serum-free culture system to evaluate the effect of different initial CD34(+) cell enrichments (Low: 24 ± 1.8%, Medium: 46 ± 2.6%, and High: 91 ± 1.5%) on the culture dynamics and outcome of HSC expansion. By combining PKH tracking dye with CD34(+) and CD34(+) CD90(+) expression, we have identified early activation of CD34 expression on CD34(-) cells in Low and Medium conditions, prior to cell division (35 ± 4.7% and 55 ± 4.1% CD34(+) cells at day 1, respectively), affecting proliferation/cell cycle status and ultimately determining CD34(+)/CD34(+) CD90(+) cell yield (High: 14 ± 1.0/3.5 ± 1.4-fold; Medium: 22 ± 2.0/3.4 ± 1,0-fold; Low: 31 ± 3.0/4.4 ± 1.5-fold) after a 7-day expansion. Considering the potential benefits of using expanded UCB HSC in transplantation, here we quantified in single UCB units, the impact of using one/two immunomagnetic sorting cycles (corresponding to Medium and High initial progenitor content), and the average CD34(+) cell recovery for each strategy, on overall CD34(+) cell expansion. The higher cell recovery upon one sorting cycle lead to higher CD34(+) cell numbers after 7 days of expansion (30 ± 2.0 vs. 13 ± 1.0 × 10(6) cells). In particular, a high (>90%) initial progenitor content was not mandatory to successfully expand HSC, since cell populations with moderate levels of enrichment readily increased CD34 expression ex-vivo, generating higher stem/progenitor cell yields. Overall, our findings stress the importance of establishing a balance between the cell proliferative potential and cell recovery upon purification, towards the efficient and cost-effective expansion of HSC for cellular therapy.

Ex vivo expansion of human mesenchymal stem cells on microcarriers.

Due to the very low titers of human mesenchymal stem cells (MSC) in their niches, namely the bone marrow, an effective approach to isolate and expand those cells ex vivo is required to meet the needs of the increasing MSC clinical applications (e.g., therapy-resistant severe acute graft-versus-host disease). Herein we describe a microcarrier-based stirred culture system protocol for the efficient ex vivo expansion of human bone marrow-derived MSC. This protocol is potentially adaptable to different culture conditions, namely focusing the use of serum-free medium formulations, other sources of MSC, or different types of microcarriers.

Gene delivery to human bone marrow mesenchymal stem cells by microporation.

Electroporation has been considered one of the most efficient non-viral based methods to deliver genes regardless of frequently observed high cell mortality. In this study we used a microporation technique to optimise the delivery of plasmid DNA encoding green fluorescence protein (GFP) to human bone marrow mesenchymal stem cells (BM-MSC). Using resuspension buffer (RB) and as low as 1.5 x 105 cells and 1 µg of DNA, we achieved 40% of cells expressing the transgene, with cell recovery and cell viabilities of 85% and 90%, respectively. An increase in DNA amount did not significantly increase the number of transfected cells but clearly reduced cell recovery. A face-centered composite design was used to unveil the conditions giving rise to optimal plasmid delivery efficiencies when using a sucrose based microporation buffer (SBB). The BM-MSC proliferation kinetics were mainly affected by the presence of plasmid and not due to the microporation process itself although no effect was observed on their immunophenotypic characteristics and differentiative potential. Based on the data shown herein microporation demonstrated to be a reliable and efficient method to genetically modify hard-to-transfect cells giving rise to the highest levels of cell survival reported so far along with superior gene delivery efficiencies.

Systematic delineation of optimal cytokine concentrations to expand hematopoietic stem/progenitor cells in co-culture with mesenchymal stem cells.

The major obstacle to the widespread use of umbilical cord blood (UCB) in hematopoietic stem/progenitor (HSC) cell therapy is the low cell dose available. A cytokine cocktail for the ex vivo expansion of UCB HSC, in co-culture with a bone marrow (BM) mesenchymal stem cells (MSC)-derived stromal layer was optimized using an experimental design approach. Proliferation of total cells (TNC), stem/progenitor cells (CD34(+)) and colony-forming units (CFU) was assessed after 7 days in culture, while sole and interactive effects of each cytokine on HSC expansion were statistically determined using a two-level Face-Centered Cube Design. The optimal cytokine cocktail obtained for HSC-MSC co-cultures was composed by SCF, Flt-3L and TPO (60, 55 and 50 ng mL(-1), respectively), resulting in 33-fold expansion in TNC, 17-fold in CD34(+) cells, 3-fold in CD34(+)CD90(+) cells and 21-fold in CFU-MIX. More importantly, these short-term expanded cells preserved their telomere length and extensively generated cobblestone area-forming cells (CAFCs) in vitro. The statistical tools used herein contributed for the rational delineation of the cytokine concentration range, in a cost-effective way, while systematically addressing complex cytokine-to-cytokine interactions, for the efficient HSC expansion towards the generation of clinically significant cell numbers for transplantation.

Maximizing the ex vivo expansion of human mesenchymal stem cells using a microcarrier-based stirred culture system.

Bioreactor systems have been developed as alternatives to standard culture flasks due to their homogeneous nature, easiness of monitoring and increased cell production. Here we investigated the in vitro expansion of bone marrow (BM) mesenchymal stem cells (MSC) in spinner flasks, using gelatin microcarriers (Cultispher S) to support cell adhesion and proliferation. MSC expansion was performed using a low-serum containing medium (2% of fetal bovine serum, FBS). A strategy was defined for the maximization of cell expansion: microcarriers were pre-coated with FBS in order to increase cell seeding efficiency and an adequate feeding regime was established (25% medium exchange everyday). The maximum cell density, 4.2 x 10(5)cells/mL, was obtained at day 8, corresponding to a fold increase in total cell number of 8.4+/-0.8. Expanded MSC retained their differentiation potential into adipogenic and osteogenic lineages, as well as their clonogenic ability. Harvested cells expressed >90% of CD73, CD90 and CD105 markers. These results demonstrated that a microcarrier-based stirred culture system is adequate for human MSC expansion, using a low-serum containing medium, allowing the generation of significant cell numbers for potential applications in regenerative medicine.

Dynamic cell-cell interactions between cord blood haematopoietic progenitors and the cellular niche are essential for the expansion of CD34+, CD34+CD38- and early lymphoid CD7+ cells.

Most clinical applications of haematopoietic stem/progenitor cells (HSCs) would benefit from their ex vivo expansion to obtain a therapeutically significant amount of cells from the available donor samples. We studied the impact of cellular interactions between umbilical cord blood (UCB) haematopoietic cells and bone marrow (BM)-derived mesenchymal stem cells (MSCs) on the ex vivo expansion and differentiative potential of UCB CD34(+)-enriched cells. UCB cells were cultured: (a) directly in contact with BM MSC-derived stromal layers (contact); (b) separated by a microporous membrane (non-contact); or (c) without stroma (no stroma). Highly dynamic culture events occurred in HSC-MSC co-cultures, involving cell-cell interactions, which preceded HSC expansion. Throughout the time in culture [18 days], total cell expansion was significantly higher in contact (fold increase of 280 + or - 37 at day 18) compared to non-contact (85 + or - 25). No significant cell expansion was observed in stroma-free cultures. CD34(+) cell expansion was also clearly favoured by direct contact with BM MSCs (35 + or - 5- and 7 + or - 3-fold increases at day 18 for contact and non-contact, respectively). Moreover, a higher percentage of CD34(+)CD38(-) cells was consistently maintained during the time in culture under contact (8.1 + or - 1.9% at day 18) compared to non-contact (5.7 + or - 1.6%). Importantly, direct cell interaction with BM MSCs significantly enhanced the expansion of early lymphoid CD7(+) cells, yielding considerably higher (x3-10) progenitor numbers compared to non-contact conditions. These results highlight the importance of dynamic cell-cell interactions between UCB HSCs and BM MSCs, towards the maximization of HSC expansion ex vivo to obtain clinically relevant cell numbers for multiple settings, such as BM transplantation or somatic cell gene therapy.

Ex vivo expansion of human mesenchymal stem cells: a more effective cell proliferation kinetics and metabolism under hypoxia.

The low bone marrow (BM) MSC titers demand a fast ex vivo expansion process to meet the clinically relevant cell dosage. Attending to the low oxygen tension of BM in vivo, we studied the influence of hypoxia on human BM MSC proliferation kinetics and metabolism. Human BM MSC cultured under 2% (hypoxia) and 20% O(2) (normoxia) were characterized in terms of proliferation, cell division kinetics and metabolic patterns. BM MSC cultures under hypoxia displayed an early start of the exponential growth phase, and cell numbers obtained at each time point throughout culture were consistently higher under low O(2), resulting in a higher fold increase after 12 days under hypoxia (40 +/- 10 vs. 30 +/- 6). Cell labeling with PKH26 allowed us to determine that after 2 days of culture, a significant higher cell number was already actively dividing under 2% compared to 20% O(2) and BM MSC expanded under low oxygen tension displayed consistently higher percentages of cells in the latest generations (generations 4-6) until the 5th day of culture. Cells under low O(2) presented higher specific consumption of nutrients, especially early in culture, but with lower specific production of inhibitory metabolites. Moreover, 2% O(2) favored CFU-F expansion, while maintaining BM MSC characteristic immunophenotype and differentiative potential. Our results demonstrated a more efficient BM MSC expansion at 2% O(2), compared to normoxic conditions, associated to an earlier start of cellular division and supported by an increase in cellular metabolism efficiency towards the maximization of cell yield for application in clinical settings.