Karen Bieback

Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue

Mesenchymal stem cells (MSCs) represent a promising tool for new clinical concepts in supporting cellular therapy. Bone marrow (BM) was the first source reported to contain MSCs. However, for clinical use, BM may be detrimental due to the highly invasive donation procedure and the decline in MSC number and differentiation potential with increasing age. More recently, umbilical cord blood (UCB), attainable by a less invasive method, was introduced as an alternative source for MSCs. Another promising source is adipose tissue (AT). We compared MSCs derived from these sources regarding morphology, the success rate of isolating MSCs, colony frequency, expansion potential, multiple differentiation capacity, and immune phenotype. No significant differences concerning the morphology and immune phenotype of the MSCs derived from these sources were obvious. Differences could be observed concerning the success rate of isolating MSCs, which was 100% for BM and AT, but only 63% for UCB. The colony frequency was lowest in UCB, whereas it was highest in AT. However, UCB‐MSCs could be cultured longest and showed the highest proliferation capacity, whereas BM‐MSCs possessed the shortest culture period and the lowest proliferation capacity. Most strikingly, UCB‐MSCs showed no adipogenic differentiation capacity, in contrast to BM‐ and AT‐MSCs. Both UCB and AT are attractive alternatives to BM in isolating MSC: AT as it contains MSCs at the highest frequency and UCB as it seems to be expandable to higher numbers.

Critical parameters for the isolation of mesenchymal stem cells from umbilical cord blood.

Evidence has emerged that mesenchymal stem cells (MSCs) represent a promising population for supporting new clinical concepts in cellular therapy. However, attempts to isolate MSCs from umbilical cord blood (UCB) of full‐term deliveries have previously either failed or been characterized by a low yield. We investigated whether cells with MSC characteristics and multi‐lineage differentiation potential can be cultivated from UCB of healthy newborns and whether yields might be maximized by optimal culture conditions or by defining UCB quality criteria.

Human Alternatives to Fetal Bovine Serum for the Expansion of Mesenchymal Stromal Cells from Bone Marrow

Mesenchymal stromal cells (MSCs) are promising candidates for novel cell therapeutic applications. For clinical scale manufacturing, human factors from serum or platelets have been suggested as alternatives to fetal bovine serum (FBS). We have previously shown that pooled human serum (HS) and thrombin‐activated platelet releasate in plasma (tPRP) support the expansion of adipose tissue‐derived MSCs. Contradictory results with bone marrow (BM)‐derived MSCs have initiated a comprehensive comparison of HS, tPRP, and pooled human platelet lysate (pHPL) and FBS in terms of their impact on MSC isolation, expansion, differentiation, and immunomodulatory activity. In addition to conventional Ficoll density gradient centrifugation, depletion of lineage marker expressing cells (RosetteSep) and CD271+ sorting were used for BM‐MSC enrichment. Cells were cultured in medium containing either 10% FBS, HS, tPRP, or pHPL. Colony‐forming units and cumulative population doublings were determined, and MSCs were maximally expanded. Although both HS and tPRP comparable to FBS supported isolation and expansion, pHPL significantly accelerated BM‐MSC proliferation to yield clinically relevant numbers within the first two passages. MSC quality and functionality including cell surface marker expression, adipogenic and osteogenic differentiation, and immunosuppressive action were similar in MSCs from all culture conditions. Importantly, spontaneous cell transformation was not observed in any of the culture conditions. Telomerase activity was not detected in any of the cultures at any passage. In contrast to previous data from adipose tissue‐derived MSCs, pHPL was found to be the most suitable FBS substitute in clinical scale BM‐MSC expansion. STEM CELLS 2009;27:2331–2341

Human AB Serum and Thrombin‐Activated Platelet‐Rich Plasma Are Suitable Alternatives to Fetal Calf Serum for the Expansion of Mesenchymal Stem Cells from Adipose Tissue

MSCs are currently in focus regarding their clinical potential in cell therapy and tissue engineering. However, most isolation and expansion protocols for clinical‐scale production of MSCs use fetal calf serum (FCS) as a supplement, which poses a potential risk for infections as well as immunological reactions. To find a suitable FCS substitute, we investigated the effects of pooled human AB serum (AB‐HS) and thrombin‐activated platelet‐rich plasma (tPRP) on adipose tissue MSCs (AT‐MSCs) with FCS as the standard control medium. AT‐MSCs of 10 donors were cultured under three different conditions: (a) 10% FCS, (b) 10% AB‐HS, and (c) 10% tPRP. Colony‐forming units, cumulative population doubling rates, and differentiation capacity toward the adipogenic and osteogenic lineages were assessed, along with immunophenotype. We demonstrated that AB‐HS and tPRP provide a significantly higher proliferative effect on AT‐MSCs than does FCS. In the first six passages, AB‐HS and tPRP MSCs exhibited a fold expansion of 66.6 ± 15.7 and 68.1 ± 6.7, respectively, compared with 24.4 ± 0.7 for FCS. Differentiation capacity was preserved throughout long‐term culture. Immunophenotype was characteristic for MSCs and comparable for all culture conditions with the exception of a distinct CD45‐/CD14‐positive side population for AB‐HS and tPRP that tended to diminish with prolonged culture. We showed that pooled human AB serum and thrombin‐activated platelet‐rich plasma are alternatives to FCS for AT‐MSCs. These human sources are better characterized regarding potential infectious threats, while providing a higher proliferation rate and retaining differentiation capacity and mesenchymal stem cell marker expression throughout long‐term culture.

Is the intravascular administration of mesenchymal stem cells safe?: Mesenchymal stem cells and intravital microscopy

We investigated the kinetics of human mesenchymal stem cells (MSCs) after intravascular administration into SCID mouse cremaster vasculature by intravital microscopy. MSCs were injected into abdominal aorta through left femoral artery at two different concentrations (1 x 10(6) or 0.2 x 10(6) cell). Arterial blood velocity decrease by 60 and 18% 1 min after high/low dose MSCs injection respectively. The blood microcirculation was interrupted after 174+/-71 and 485+/-81 s. Intravital microscopy observation and histopathologic analysis of cremaster muscles indicated MSCs were entrapped in capillaries in both groups. 40 and 25% animals died of pulmonary embolism respectively in both high and low MSCs dose groups, which was detected by histopathologic analysis of the lungs. Intraarterial MSCs administration may lead to occlusion in the distal vasculature due to their relatively large cell size. Pulmonary sequestration may cause death in small laboratory animals. MSCs should be used cautiously for intravascular transplantation.

Phenotype, donor age and gender affect function of human bone marrow-derived mesenchymal stromal cells

BackgroundMesenchymal stromal cells (MSCs) are attractive for cell-based therapies ranging from regenerative medicine and tissue engineering to immunomodulation. However, clinical efficacy is variable and it is unclear how the phenotypes defining bone marrow (BM)-derived MSCs as well as donor characteristics affect their functional properties.MethodsBM-MSCs were isolated from 53 (25 female, 28 male; age: 13 to 80 years) donors and analyzed by: (1) phenotype using flow cytometry and cell size measurement; (2) in vitro growth kinetics using population doubling time; (3) colony formation capacity and telomerase activity; and (4) function by in vitro differentiation capacity, suppression of T cell proliferation, cytokines and trophic factors secretion, and hormone and growth factor receptor expression. Additionally, expression of Oct4, Nanog, Prdm14 and SOX2 mRNA was compared to pluripotent stem cells.ResultsBM-MSCs from younger donors showed increased expression of MCAM, VCAM-1, ALCAM, PDGFRβ, PDL-1, Thy1 and CD71, and led to lower IL-6 production when co-cultured with activated T cells. Female BM-MSCs showed increased expression of IFN-γR1 and IL-6β, and were more potent in T cell proliferation suppression. High-clonogenic BM-MSCs were smaller, divided more rapidly and were more frequent in BM-MSC preparations from younger female donors. CD10, β1integrin, HCAM, CD71, VCAM-1, IFN-γR1, MCAM, ALCAM, LNGFR and HLA ABC were correlated to BM-MSC preparations with high clonogenic potential and expression of IFN-γR1, MCAM and HLA ABC was associated with rapid growth of BM-MSCs. The mesodermal differentiation capacity of BM-MSCs was unaffected by donor age or gender but was affected by phenotype (CD10, IFN-γR1, GD2). BM-MSCs from female and male donors expressed androgen receptor and FGFR3, and secreted VEGF-A, HGF, LIF, Angiopoietin-1, basic fibroblast growth factor (bFGF) and NGFB. HGF secretion correlated negatively to the expression of CD71, CD140b and Galectin 1. The expression of Oct4, Nanog and Prdm14 mRNA in BM-MSCs was much lower compared to pluripotent stem cells and was not related to donor age or gender. Prdm14 mRNA expression correlated positively to the clonogenic potential of BM-MSCs.ConclusionsBy identifying donor-related effects and assigning phenotypes of BM-MSC preparations to functional properties, we provide useful tools for assay development and production for clinical applications of BM-MSC preparations.

Mesenchymal Stromal Cells from Umbilical Cord Blood

Mesenchymal Stromal Cells (MSC) are key candidates for cellular therapies. Although most therapeutic applications have focused on adult bone marrow derived MSC, increasing evidence suggests that MSC are present within a wide range of tissues. Umbilical cord blood (CB) has been proven to be a valuable source of hematopoietic stem cells, but its therapeutic potential extends beyond the hematopoietic component suggesting regenerative potential in solid organs as well. There is evidence that other stem or progenitor populations, such as MSC, exist in CB which might be responsible for these effects. Many different stem and progenitor cell populations have been postulated with potential ranging from embryonic like to lineage-committed progenitor cells. Based on the confusing data, this review focuses on a human CB derived, plastic adherent fibroblastoid population expressing similar characteristics to bone marrow derived MSC. It concentrates especially on concepts of isolation and expansion, comparing the phenotype with bone marrow derived MSC, describing the differentiation capacity and finally in the last the therapeutic potential with regard to regenerative medicine, stromal support, immune modulation and gene therapy.

Cell Origin of Human Mesenchymal Stem Cells Determines a Different Healing Performance in Cardiac Regeneration

The possible different therapeutic efficacy of human mesenchymal stem cells (hMSC) derived from umbilical cord blood (CB), adipose tissue (AT) or bone marrow (BM) for the treatment of myocardial infarction (MI) remains unexplored. This study was to assess the regenerative potential of hMSC from different origins and to evaluate the role of CD105 in cardiac regeneration. Male SCID mice underwent LAD-ligation and received the respective cell type (400.000/per animal) intramyocardially. Six weeks post infarction, cardiac catheterization showed significant preservation of left ventricular functions in BM and CD105+-CB treated groups compared to CB and nontreated MI group (MI-C). Cell survival analyzed by quantitative real time PCR for human GAPDH and capillary density measured by immunostaining showed consistent results. Furthermore, cardiac remodeling can be significantly attenuated by BM-hMSC compared to MI-C. Under hypoxic conditions in vitro, remarkably increased extracellular acidification and apoptosis has been detected from CB-hMSC compared to BM and CD105 purified CB-derived hMSC. Our findings suggests that hMSC originating from different sources showed a different healing performance in cardiac regeneration and CD105+ hMSC exhibited a favorable survival pattern in infarcted hearts, which translates into a more robust preservation of cardiac function.

Mesenchymal stem cells and cardiac repair

•  Introduction •  MSC isolation, characterization and standardization ‐  Isolation from different sources ‐  Isolation under different culture conditions ‐  Characterization of MSCs ‐  Standardization of MSCs •  Mechanisms of cardiac repair ‐  Differentiation of MSCs towards cardiomyocytes ‐  Paracrine effect of MSCs ‐  MSCs and blood vessel regeneration ‐  MSC integration into the injured myocardium •  Ex vivo manipulation of MSCs ‐  Pre‐treatment with growth factors ‐  Genetic engineering ‐  Hypoxia preconditioning ‐  Pharmacological interventions •  Pre‐clinical application on cardiovascular disease •  Clinical application: where are we? •  Summary

Platelet Lysate as Replacement for Fetal Bovine Serum in Mesenchymal Stromal Cell Cultures

Mesenchymal stromal cells (MSC) emerged as highly attractive in cell-based regenerative medicine. Initially thought to provide cells capable of differentiation towards mesenchymal cell types (osteoblasts, chondrocytes, adipocytes etc.), by and by potent immunoregulatory and pro-regenerative activities have been discovered, broadening the field of potential applications from bone and cartilage regeneration to wound healing and treatment of autoimmune diseases. Due to the limited frequency in most tissue sources, ex vivo expansion of MSC is required compliant with good manufacturing practice (GMP) guidelines to yield clinically relevant cell doses. Though, still most manufacturing protocols use fetal bovine serum (FBS) as cell culture supplement to isolate and to expand MSC. However, the high lot-to-lot variability as well as risk of contamination and immunization call for xenogenic-free culture conditions. In terms of standardization, chemically defined media appear as the ultimate achievement. Since these media need to maintain all key cellular and therapy-relevant features of MSC, the development of chemically defined media is still - albeit highly investigated - only in its beginning. The current alternatives to FBS rely on human blood-derived components: plasma, serum, umbilical cord blood serum, and platelet derivatives like platelet lysate. Focusing on quality aspects, the latter will be addressed within this review.