The therapeutic potential of bone marrow-derived stromal cells

Since the replacement of the hematopoietic system became feasible through bone marrow (BM) transplantation, the idea of how to replace other organs of the body has been in the forefront of medical research. Scientists have been searching for the ideal stem cell that could be manipulated to differentiate into any tissue. Although the embryonal stem cells seemed to have the ability to do this, the difficulties surrounding their use prevented them from becoming therapeutically useful. Thus, the field turned to adult stem cells, particularly stem cells of BM origin. We have learnt a lot during the last decade about the potential of the BM-derived stromal (also called mesenchymal stem) cells (BMSCs). The first studies suggested them as cell replacement tools, but later it turned out that their usefulness is more likely due to paracrine effects due to a large variety of secreted factors that induce growth and differentiation of the tissue-specific stem cells as well as prevent injured cells from apoptotic death. Finally, a whole new field emerged when many groups confirmed that these cells are also capable of regulating immune function in a so far unknown, dynamic manner. When BMSCs are injected they seem to be able to sense the environment and respond according to the actual need of the organism in order to survive. This plasticity can never be done by the use of any drugs and such a "live" cell therapy could open a whole new chapter in clinical care in the future.     

Impact of bone marrow-derived mesenchymal stromal cells on experimental xenogeneic graft-versus-host disease

Background aimsGraft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation caused by donor T cells reacting against host tissues. Previous studies have suggested that mesenchymal stromal cells (MSCs) could exert potent immunosuppressive effects.MethodsThe ability of human bone marrow derived MSCs to prevent xenogeneic GVHD in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice and in NOD/SCID/interleukin-2Rγ(null) (NSG) mice transplanted with human peripheral blood mononuclear cells (PBMCs) was assessed.ResultsInjection of 200 × 10⁶ human PBMCs intraperitoneally (IP) into sub-lethally (3.0 Gy) irradiated NOD/SCID mice also given anti-asialo GM₁ antibodies IP 1 day prior and 8 days after transplantation induced lethal xenogeneic GVHD in all tested mice. Co-injection of 2 × 10⁶ MSCs IP on day 0 did not prevent lethal xenogeneic GVHD induced by injection of human PBMCs. Similarly, injection of 30 × 10⁶ human PBMCs IP into sub-lethally (2.5 Gy) irradiated NSG mice induced a lethal xenogeneic GVHD in all tested mice. Injection of 3 × 10⁶ MSCs IP on days 0, 7, 14 and 21 did not prevent lethal xenogeneic GVHD induced by injection of human PBMCs.ConclusionsInjection of MSCs did not prevent xenogeneic GVHD in these two humanized mice models.     

Bone marrow stromal cells (bone marrow-derived multipotent mesenchymal stromal cells) for bone tissue engineering: basic science to clinical translation

Bone tissue engineering is a promising field of regenerative medicine in which cultured cells, scaffolds, and osteogenic inductive signals are used to regenerate bone. This technology has already been used in several clinical studies and its efficacy has been reported. In this review, we focus on bone marrow stromal cells, which are the most commonly used cell source for bone tissue engineering. The nature of the cells, suitable culture conditions for bone tissue engineering, and their potential therapeutic applications are reviewed with possible caveats. Furthermore, recent advances in bone marrow stromal cell biology are discussed with reference to clinical translation.     

Directing bone marrow-derived stromal cell function with mechanics

Because bone marrow-derived stromal cells (BMSCs) are able to generate many cell types, they are envisioned as source of regenerative cells to repair numerous tissues, including bone, cartilage, and ligaments. Success of BMSC-based therapies, however, relies on a number of methodological improvements, among which better understanding and control of the BMSC differentiation pathways. Since many years, the biochemical environment is known to govern BMSC differentiation, but more recent evidences show that the biomechanical environment is also directing cell functions. Using in vitro systems that aim to reproduce selected components of the in vivo mechanical environment, it was demonstrated that mechanical loadings can affect BMSC proliferation and improve the osteogenic, chondrogenic, or myogenic phenotype of BMSCs. These effects, however, seem to be modulated by parameters other than mechanics, such as substrate nature or soluble biochemical environment. This paper reviews and discusses recent experimental data showing that despite some knowledge limitation, mechanical stimulation already constitutes an additional and efficient tool to drive BMSC differentiation.     

Isolation, characterization, and chondrogenic potential of human bone marrow-derived multipotential stromal cells

Multipotential bone marrow stromal cells have the ability to differentiate along multiple connective tissue lineages including cartilage. In this study, we developed an efficient and reproducible procedure for the isolation of stromal cells from bone marrow aspirates of normal human donors based on the expression of endoglin, a type III receptor of the transforming growth factor-beta (TGF-beta) receptor family. We demonstrate that these cells have the ability of multiple lineage differentiation. Stromal cells represented 2-3% of the total mononuclear cells of the marrow. The cells displayed a fibroblastic colony formation in monolayer culture and maintained similar morphology with passage. Expression of cell surface molecules by flow cytometry displayed a stable phenotype with culture expansion. When cocultured with hematopoietic CD34(+) progenitor cells, stromal cells were able to maintain their ability to support hematopoiesis in vitro. Culture expanded stromal cells were placed in a 3-dimensional matrix of alginate beads and cultured in serum-free media in the presence of TGFbeta-3 for chondrogenic lineage progression. Increased expression of type II collagen messenger RNA was observed in the TGFbeta3 treated cultures. Immunohistochemistry performed on sections of alginate beads detected the presence of type II collagen protein. This isolation procedure for stromal cells and the establishment of the alginate culture system for chondrogenic progression will contribute to the understanding of chondrogenesis and cartilage repair.     

Isolation,expansion and characterization of bone marrow-derived mesenchymal stromal cells in serum-free conditions

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) heralded a new beginning for regenerative medicine and generated tremendous interest as the most promising source for therapeutic application. Most cell therapies require stringent regulatory compliance and prefer the use of serum-free media (SFM) or xeno-free media (XFM) for the MSC production process, starting from the isolation onwards. Here, we report on serum-free isolation and expansion of MSCs and compare them with cells grown in conventional fetal bovine serum (FBS)-containing media as a control. The isolation, proliferation and morphology analysis demonstrated significant differences between MSCs cultured in various SFM/XFM in addition to their difference with FBS controls. BD Mosaic? Mesenchymal Stem Cell Serum-Free media (BD-SFM) and Mesencult-XF (MSX) supported the isolation, sequential passaging, tri-lineage differentiation potential and acceptable surface marker expression profile of BM-MSCs. Further, MSCs cultured in SFM showed higher immune suppression and hypo-immunogenicity properties, making them an ideal candidate for allogeneic cell therapy. Although cells cultured in control media have a significantly higher proliferation rate, BM-MSCs cultured in BD-SFM or MSX media are the preferred choice to meet regulatory requirements as they do not contain bovine serum. While BM-MSCs cultured in BD-SFM and MSX media adhered to all MSC characteristics, in the case of few parameters, the performance of cells cultured in BD-SFM was superior to that of MSX media. Pre-clinical safety and efficiency studies are required before qualifying SFM or XFM media-derived MSCs for therapeutic applications.     

Genetic stability of bone marrow-derived human mesenchymal stromal cells in the Quantum System

Background aimsThe Quantum® Cell Expansion System (Quantum; Terumo BCT, Inc, Lakewood, CO, USA) is a novel hollow fiber-based device that automates and closes the cell culture process, reducing labor intensive tasks such as manual cell culture feeding and harvesting. The manual cell selection and expansion processes for the production of clinical-scale quantities of bone marrow-derived human mesenchymal stromal cells (BM-hMSCs) have been successfully translated onto the Quantum platform previously. The formerly static, manual, in vitro process performed primarily on tissue culture polystyrene substrates may raise the question of whether BM-hMSCs cultured on a hollow fiber platform yields comparable cell quality.MethodsA rigorous battery of assays was used to determine the genetic stability of BM-hMSCs selected and produced with the Quantum. In this study, genetic stability was determined by assessing spectral karyotype, micronucleus formation and tumorigenicity to resolve chromosomal aberrations in the stem cell population. Cell phenotype, adherent growth kinetics and tri-lineage differentiation were also evaluated. HMSC bone marrow aspirates, obtained from three approved donors, were expanded in parallel using T225 culture flasks and the Quantum.ResultsBM-hMSCs harvested from the Quantum demonstrated immunophenotype, morphology and tri-lineage differentiation capacity characteristics consistent with the International Society of Cell Therapy standard for hMSCs. Cell populations showed no malignant neoplastic formation in athymic mice 60 days post-transplant, no clonal chromosomal aberrations were observed and no DNA damage was found as measured by micronucleus formation.ConclusionsQuantum-produced BM-hMSCs are of comparable quality and demonstrate analogous genetic stability to BM-hMSCs cultured on tissue culture polystyrene substrates.     

Chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells in a three-dimensional environment

Cell therapy combined with biomaterial scaffolds is used to treat cartilage defects. We hypothesized that chondrogenic differentiation bone marrow-derived mesenchymal stem cells (BM-MSCs) in three-dimensional biomaterial scaffolds would initiate cartilaginous matrix deposition and prepare the construct for cartilage regeneration in situ. The chondrogenic capability of human BM-MSCs was first verified in a pellet culture. The BM-MSCs were then either seeded onto a composite scaffold rhCo-PLA combining polylactide and collagen type II (C2) or type III (C3), or commercial collagen type I/III membrane (CG). The BM-MSCs were either cultured in a proliferation medium or chondrogenic culture medium. Adult human chondrocytes (ACs) served as controls. After 3, 14, and 28 days, the constructs were analyzed with quantitative polymerase chain reaction and confocal microscopy and sulfated glycosaminoglycans (GAGs) were measured. The differentiated BM-MSCs entered a hypertrophic state by Day 14 of culture. The ACs showed dedifferentiation with no expression of chondrogenic genes and low amount of GAG. The CG membrane induced the highest expression levels of hypertrophic genes. The two different collagen types in composite scaffolds yielded similar results. Regardless of the biomaterial scaffold, culturing BM-MSCs in chondrogenic differentiation medium resulted in chondrocyte hypertrophy. Thus, caution for cell fate is required when designing cell-biomaterial constructs for cartilage regeneration.     

Sequestration and homing of bone marrow-derived lineage negative progenitor cells in the lung during pneumococcal pneumonia

Background

Bone marrow (BM)-derived progenitor cells have been shown to have the potential to differentiate into a diversity of cell types involved in tissue repair. The characteristics of these progenitor cells in pneumonia lung is unknown. We have previously shown that Streptococcus pneumoniae induces a strong stimulus for the release of leukocytes from the BM and these leukocytes preferentially sequester in the lung capillaries. Here we report the behavior of BM-derived lineage negative progenitor cells (Lin- PCs) during pneumococcal pneumonia using quantum dots (QDs), nanocrystal fluorescent probes as a cell-tracking technique.

Methods

Whole BM cells or purified Lin- PCs, harvested from C57/BL6 mice, were labeled with QDs and intravenously transfused into pneumonia mice infected by intratracheal instillation of Streptococcus pneumoniae. Saline was instilled for control. The recipients were sacrificed 2 and 24 hours following infusion and QD-positive cells retained in the circulation, BM and lungs were quantified.

Results

Pneumonia prolonged the clearance of Lin- PCs from the circulation compared with control (21.7 ± 2.7% vs. 7.7 ± 0.9%, at 2 hours, P < 0.01), caused preferential sequestration of Lin- PCs in the lung microvessels (43.3 ± 8.6% vs. 11.2 ± 3.9%, at 2 hours, P < 0.05), and homing of these cells to both the lung (15.1 ± 3.6% vs. 2.4 ± 1.2%, at 24 hours, P < 0.05) and BM as compared to control (18.5 ± 0.8% vs. 9.5 ± 0.4%, at 24 hours, P < 0.01). Very few Lin- PCs migrated into air spaces.

Conclusion

In this study, we demonstrated that BM-derived progenitor cells are preferentially sequestered and retained in pneumonic mouse lungs. These cells potentially contribute to the repair of damaged lung tissue.     

Hydroxyapatite/regenerated silk fibroin scaffold-enhanced osteoinductivity and osteoconductivity of bone marrow-derived mesenchymal stromal cells

A novel hydroxyapatite/regenerated silk fibroin scaffold was prepared and investigated for its potential to enhance both osteoinductivity and osteoconductivity of bone marrow-derived mesenchymal stromal cells in vitro. Approx. 12.4 ± 0.06 % (w/w) hydroxyapatite was deposited onto the scaffold, and cell viability and DNA content were significantly increased (18.5 ± 0.6 and 33 ± 1.2 %, respectively) compared with the hydroxyapatite scaffold after 14 days. Furthermore, alkaline phosphatase activity in the novel scaffold increased 41 ± 2.5 % after 14 days compared with the hydroxyapatite scaffold. The data indicate that this novel hydroxyapatite/regenerated silk fibroin scaffold has a positive effect on osteoinductivity and osteoconductivity, and may be useful for bone tissue engineering.     

A robust and reproducible animal serum-free culture method for clinical-grade bone marrow-derived mesenchymal stromal cells

Efficient xenofree expansion methods to replace fetal bovine serum (FBS)-based culture methods are strongly encouraged by the regulators and are needed to facilitate the adoption of mesenchymal stromal cell (MSC)-based therapies. In the current study we established a clinically-compliant and reproducible animal serum-free culture protocol for bone marrow-(BM-) MSCs based on an optimized platelet-derived supplement. Our study compared two different platelet-derived supplements, platelet lysate PL1 versus PL2, produced by two different methods and lysed with different amounts of freeze–thaw cycles. Our study also explored the effect of a low oxygen concentration on BM-MSCs. FBS-supplemented BM-MSC culture served as control. Growth kinetics, differentiation and immunomodulatory potential, morphology, karyotype and immunophenotype was analysed. Growth kinetics in long-term culture was also studied. Based on the initial results, we chose to further process develop the PL1-supplemented culture protocol at 20 % oxygen. The results from 11 individual BM-MSC batches expanded in the chosen condition were consistent, yielding 6.60 × 10⁹ ± 4.74 × 10⁹ cells from only 20 ml of bone marrow. The cells suppressed T-cell proliferation, displayed normal karyotype and typical MSC differentiation potential and phenotype. The BM-MSCs were, however, consistently HLA-DR positive when cultured in platelet lysate (7.5–66.1 %). We additionally show that culture media antibiotics and sterile filtration of the platelet lysate can be successfully omitted. We present a robust and reproducible clinically-compliant culture method for BM-MSCs based on platelet lysate, which enables high quantities of HLA-DR positive MSCs at a low passage number (p2) and suitable for clinical use.     

Taurine postponed the replicative senescence of rat bone marrow-derived multipotent stromal cells in vitro

The aging of many mammalian tissues is associated with replicative decline in somatic stem cells. Postponing this decline is a direct way of anti-aging. Bone marrow-derived multipotent stromal cells (BMSCs) hold promise for an increasing list of therapeutic uses due to their multilineage potential. Clinical application of BMSCs requires abundant cells that can be overcome by ex vivo expansion of cells, but often facing the replicative senescence problem. We demonstrated that taurine exhibited anti-replicative senescence effect on rat BMSCs by promoting colony forming unit-fibroblast formation and cell proliferation, shortening cell population doubling time, enormously inhibiting senescence-associated beta-galactosidase activity and slowing the loss of differentiation potential, while having no significant effect on the maximum passage number and total culture time, and slight influences on the cell surface CD molecules expressions. Taurine is a quite safe antioxidant and nutrient extensively used in food addition and clinical treatment. These suggested that taurine is a promising anti-replicative senescence additive for ex vivo expansion of BMSCs in experimental and clinical cell therapies.     

Cytokine production by M-CSF- and GM-CSF-induced mouse bone marrow-derived macrophages upon coculturing with late apoptotic cells

Recently, we found that resident peritoneal macrophages produce MIP-2, one of the major chemokines for neutrophils, upon coculturing with late apoptotic cells, and that intraperitoneal injection of late apoptotic cells into the peritoneal cavity causes neutrophil infiltration via MIP-2. It is not known, however, whether or not macrophages are heterogeneous in such MIP-2 production. In this study, we examined changes in the surface phenotype during the differentiation of bone marrow cells into macrophages due to M-CSF and GM-CSF, and then examined the production of cytokines, namely IL-12 p40, MIP-2, IL-10, and TGF-β, following phagocytosis of late apoptotic cells with these macrophages or LPS stimulation of these macrophages. GM-CSF and M-CSF induced macrophage populations with distinct but overlapping cell surface phenotype. Although these macrophages phagocytosed late apoptotic cells to a similar extent, they produced either IL-12 p40 or IL-10, whereas they produced MIP-2 to a similar extent after the coculture, raising the possibility that late apoptotic cells may induce neutrophil infiltration in any organs, such as the liver and lungs, where the macrophages are differentiated by either M-CSF or GM-CSF, respectively.     

Activated T cells modulate immunosuppression by embryonic-and bone marrow-derived mesenchymal stromal cells through a feedback mechanism

Background aimsHuman embryonic stem cell (hESC)-derived mesenchymal stromal cells (MSC) (hESC-MSC) are an alternative source of MSC to bone marrow (BM)-derived MSC (BM-MSC), which are being investigated in clinical trials for their immunomodulatory potential. hESC-MSC have the advantage of being consistent because each batch can be generated from hESC under defined conditions. In contrast, BM-MSC have a limited proliferative capacity.MethodsThe ability to suppress the proliferation of anti-CD3/CD28-stimulated CD4 ⁺ T cells by hESC-MSC was compared with adult BM-MSC and neonatal foreskin fibroblast (Fb).ResultshESC-MSC suppress the proliferation of CD4 ⁺ T cells in both contact and transwell systems, although inhibition is less in the transwell system. hESC-MSC are approximately 2-fold less potent (67 cells/100 T cells) than BM-MSC and Fb (37 and 34 cells/100 T cells, respectively) at suppressing T-cell proliferation by 50% in a transwell [inhibitory concentration(IC)₅₀]. The anti-proliferative effect is not contact-dependent but requires the presence of factors such as interferon (IFN)-γ produced by activated T cells. IFN-γ induces the expression of indoleamine-2,3-dioxygenase (IDO) in hESC-MSC, BM-MSC and Fb, contributing to their immunosuppressive property.ConclusionsThe feedback loop between MSC or Fb and activated T cells may limit the immunosuppressive effects of MSC and Fb to sites containing ongoing immunologic or inflammatory responses where activated T cells induce the up-regulation of IDO and immunomodulatory properties of MSC and Fb. These data demonstrate that hESC-MSC may be evaluated further as an allogeneic cell source for therapeutic applications requiring immunosuppression.     

The role of bone marrow-derived stromal cells in the maintenance of plasma cell longevity

Protective circulating Abs originate primarily from long-lived plasma cells in the bone marrow. However, the molecular and cellular basis of plasma cell longevity is unknown. We investigated the capacity of primary bone marrow-derived stromal cells to maintain plasma cell viability in vitro. Plasma cells purified from the bone marrow or lymph nodes died rapidly when plated in media, but a subpopulation of plasma cells survived and secreted high levels of Ab for up to 4 wk when cocultured with stromal cells. Ab secretion was inhibited by the addition of anti-very late Ag-4 to plasma cell/stromal cell cocultures indicating that direct interactions occur and are necessary between stromal cells and plasma cells. The addition of rIL-6 to plasma cells cultured in media alone partially relieved the sharp decline in Ab secretion observed in the absence of stromal cells. Moreover, when stromal cells from IL-6(-/-) mice were used in plasma cell/stromal cell cocultures, Ab levels decreased 80% after 7 days as compared with wild-type stromal cells. Further, IL-6 mRNA message was induced in stromal cells by coculture with plasma cells. These data indicate that bone marrow plasma cells are not intrinsically long-lived, but rather that plasma cells contact and modify bone marrow stromal cells to provide survival factors.