Flow Cytometric Characterization of Hematopoietic Stem and Progenitor Cell Subpopulations in Autologous Peripheral Blood Stem Cell Preparations after Cryopreservation.

Introduction: Autologous stem cell transplantation is a successful routine procedure with only a small number of non-engraftment cases, although the time to hematopoietic recovery may vary considerably across patients. While CD34 has been the decisive marker for enumerating hematopoietic stem and progenitor cells (HSPCs) for more than 30 years, the impact of CD34-positive cellular subpopulations in autologous HSPC grafts on hematopoietic reconstitution remains unclear. Methods: The two-color ISHAGE protocol represents the current gold standard for CD34+ cell enumeration but includes only the number of viable CD45+/CD34+ cells relative to the body weight of the recipient. We adapted a multicolor flow cytometry marker panel for advanced characterization of CD34 subpopulations in retained samples of autologous peripheral blood stem cell products (n = 49), which had been cryostored for a wide range from 4 to 15 years. The flow cytometric analysis included CD10, CD34, CD38, CD45, CD45RA, CD133, and viability staining with 7AAD. The findings were correlated with clinical engraftment data, including reconstitution of leukocytes, neutrophils, and platelets after transplantation (TPL). Results: We demonstrated that the identification of autologous HSPC subpopulations by flow cytometry after cryopreservation is feasible. Regarding the distribution of HSPC subpopulations, a markedly different pattern was observed in comparison to previously published data obtained using fresh autologous material. Our data revealed the largest ratio of lympho-myeloid progenitors (LMPPs) after freezing and thawing, followed by multipotent progenitors and erythroid-myeloid progenitors. A high ratio of LMPPs, representing an immature stage of differentiation, correlated significantly with early neutrophilic granulocyte and leukocyte engraftment (p = 0.025 and p = 0.003). Conversely, a large ratio of differentiated cells correlated with late engraftment of neutrophilic granulocytes (p = 0.024). Overall, successful engraftment was documented for all patients. Conclusion: We established an advanced flow cytometry panel to assess the differentiation ability of cryostored autologous peripheral blood stem cell grafts and correlated it with timely hematopoietic reconstitution. This approach represents a novel and comprehensive way to identify hematopoietic stem and progenitor subpopulations. It is a feasible way to indicate the engraftment capacity of stem cell products.

Closer to Nature: The Role of MSCs in Recreating the Microenvironment of the Hematopoietic Stem Cell Niche in vitro.

Background: The stem cell niche in human bone marrow provides scaffolds, cellular frameworks and essential soluble cues to support the stemness of hematopoietic stem and progenitor cells (HSPCs). To decipher this complex structure and the corresponding cellular interactions, a number of in vitro model systems have been developed. The cellular microenvironment is of key importance, and mesenchymal stromal cells (MSCs) represent one of the major cellular determinants of the niche. Regulation of the self-renewal and differentiation of HSPCs requires not only direct cellular contact and adhesion molecules, but also various cytokines and chemokines. The C-X-C chemokine receptor type 4/stromal cell-derived factor 1 axis plays a pivotal role in stem cell mobilization and homing. As we have learned in recent years, to realistically simulate the physiological in vivo situation, advanced model systems should be based on niche cells arranged in a three-dimensional (3D) structure. By providing a dynamic rather than static setup, microbioreactor systems offer a number of advantages. In addition, the role of low oxygen tension in the niche microenvironment and its impact on hematopoietic stem cells need to be taken into account and are discussed in this review. Summary: This review focuses on the role of MSCs as a part of the bone marrow niche, the interplay between MSCs and HSPCs and the most important regulatory factors that need to be considered when engineering artificial hematopoietic stem cell niche systems. Conclusion: Advanced 3D model systems using MSCs as niche cells and applying microbioreactor-based technology are capable of simulating the natural properties of the bone marrow niche more closely than ever before.

Co-culture with mesenchymal stromal cells increases proliferation and maintenance of haematopoietic progenitor cells.

Mesenchymal stromal cells (MSC) have been suggested to provide a suitable cellular environment for in vitro expansion of haematopoietic stem and progenitor cells (HPC) from umbilical cord blood. In this study, we have simultaneously analysed the cell division history and immunophenotypic differentiation of HPC by using cell division tracking with carboxyfluorescein diacetate N-succinimidyl ester (CFSE). Co-culture with MSC greatly enhanced proliferation of human HPC, especially of the more primitive CD34(+)CD38(-) fraction. Without co-culture CD34 and CD133 expressions decreased after several cell divisions, whereas CD38 expression was up-regulated after some cell divisions and then diminished in fast proliferating cells. Co-culture with MSC maintained a primitive immunophenotype (CD34(+), CD133(+) and CD38(-)) for more population doublings, whereas up-regulation of differentiation markers (CD13, CD45 and CD56) in HPC was delayed to higher numbers of cell divisions. Especially MSC of early cell passages maintained CD34 expression in HPC over more cell divisions, whereas MSC of higher passages further enhanced their proliferation rate. Inhibition of mitogen-activated protein kinase 1 (MAPK1) impaired proliferation and differentiation of HPC, but not maintenance of long-term culture initiating cells. siRNA knockdown of N-cadherin and VCAM1 in feeder layer cells increased the fraction of slow dividing HPC, whereas knockdown of integrin beta 1 (ITGB1) and CD44 impaired their differentiation. In conclusion, MSC support proliferation as well as self-renewal of HPC with primitive immunophenotype. The use of early passages of MSC and genetic manipulation of proteins involved in HPC-MSC interaction might further enhance cord blood expansion on MSC.

Molecular and secretory profiles of human mesenchymal stromal cells and their abilities to maintain primitive hematopoietic progenitors.

Mesenchymal stromal cells (MSC) provide a supportive cellular microenvironment and are able to maintain the self-renewal capacity of hematopoietic progenitor cells (HPC). Isolation procedures for MSC vary extensively, and this may influence their biologic properties. In this study, we have compared human MSC isolated from bone marrow (BM) using two culture conditions, from cord blood (CB), and from adipose tissue (AT). The ability to maintain long-term culture-initiating cell frequency and a primitive CD34(+)CD38(-) immunophenotype was significantly higher for MSC derived from BM and CB compared with those from AT. These results were in line with a significantly higher adhesion of HPC to MSC from BM and CB versus MSC from AT. We have compared the cytokine production of MSC by cytokine antibody arrays, enzyme-linked immunosorbent assay, and a cytometric bead array. There were reproducible differences in the chemokine secretion profiles of various MSC preparations, but there was no clear concordance with differences in their potential to maintain primitive function of HPC. Global gene expression profiles of MSC preparations were analyzed and showed that adhesion proteins including cadherin-11, N-cadherin, vascular cell adhesion molecule 1, neural cell adhesion molecule 1, and integrins were highly expressed in MSC preparations derived from BM and CB. Thus, MSC from BM and CB are superior to MSC from AT for maintenance of primitive HPC. The latter property is associated with specific molecular profiles indicating the significance of cell-cell junctions but not with secretory profiles. Disclosure of potential conflicts of interest is found at the end of this article.

Adhesion of human hematopoietic progenitor cells to mesenchymal stromal cells involves CD44.

BACKGROUND: Direct cell-cell contact between hematopoietic progenitor cells (HPC) and their cellular microenvironment is essential for maintenance of 'stemness'. We have previously demonstrated that a feeder layer of human mesenchymal stromal cells (MSC) could provide a surrogate model as a niche for human HPC. Maintenance of long-term culture-initiating cells was significantly lower on fibroblasts. METHODS: Adhesion of HPC to MSC was further analyzed using our recently described adhesion assay based on gravitational force upon inversion and in combination with specific antibodies against CD44. RESULTS: Adhesion of KG1a and CD34+ cells was significantly reduced by administration of a monoclonal CD44 antibody and for KG1a to a greater extent than for CD34+ cells. Interaction of HPC and MSC was further analyzed by laser scanning confocal microscopy. CD44 was located on the uropod of CD34+ cells at the site of contact with MSC and both cell types were interwoven by a network of fibronectin. CONCLUSION: Various adhesion proteins, including CD44, are involved in the contact of human HPC and human MSC and further analysis of the relative significance and interaction of these proteins will be crucial for the understanding of the mechanism of this specific cell-cell interaction.

Evaluation of GMP-compliant culture media for in vitro expansion of human bone marrow mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) from human bone marrow serve as a resource for cell-based therapies in regenerative medicine. Clinical applications require standardized protocols according to good manufacturing practice (GMP) guidelines. Donor variability as well as the intrinsic heterogeneity of MSC populations must be taken into consideration. The composition of the culture medium is a key factor in successful MSC expansion. The aim of this study was to comparatively assess the efficiency of xeno-free human platelet lysate (HPL)-based cell expansion with two commercially available media-StemPro MSC SFM CTS (for human ex vivo tissue and cell culture processing applications) and MSCGM (non-GMP-compliant, for research only)-in an academic setting as the first optimization step toward GMP-compliant manufacturing. We report the feasibility of MSC expansion up to the yielded cell number with all three media. MSCs exhibited the typical fibroblastoid morphology, with distinct differences in cell size depending on the medium. The differentiation capacity and characteristic immunophenotype were confirmed for all MSC populations. Proliferation was highest using StemPro MSC SFM CTS, whereas HPL medium was more cost-effective and its composition could be adjusted individually according to the respective needs. In summary, we present a comprehensive evaluation of GMP-compatible culture media for MSC expansion. Both StemPro and HPL medium proved to be suitable for clinical application and allowed sufficient cell proliferation. Specific differences were observed and should be considered according to the intended use. This study provides a detailed cost analysis and tools that may be helpful for the establishment of GMP-compliant MSC expansion.

KATP channels in mesenchymal stromal stem cells: strong up-regulation of Kir6.2 subunits upon osteogenic differentiation.

The promising use of mesenchymal stromal cells (MSC) in regenerative technologies accounts for necessity of detailed study of their physiology. Proliferation and differentiation of multipotent cells often involve changes in their metabolic state. In the present study, we analyzed the expression of ATP-sensitive potassium (K(ATP)) channels in MSC and upon in vitro differentiation. K(ATP) channels are present in many cells and regulate a variety of cellular functions by coupling cell metabolism with membrane potential. Kir6.1, Kir6.2 and SUR2A were expressed in undifferentiated MSC, whereas SUR2B and SUR1 were not detected on cDNA and protein level. Upon adipogenic differentiation Kir6.1 and SUR2A showed a significant reduction of the amount of mRNA by 84% and 95%, respectively, whereas Kir6.2 expression was unchanged. Osteogenic differentiation strongly up-regulated Kir6.2 mRNA (28-fold) whereas Kir6.1 and SUR2A showed no significant change in expression. Quantitative Western blot analysis and immunofluorescence staining confirmed the elevated expression of Kir6.2 upon osteogenic differentiation. Taken together, expression changes of K(ATP) channels may contribute to in vitro differentiation of MSC and represent changes in the metabolic state of the developing tissue.

N-cadherin is expressed on human hematopoietic progenitor cells and mediates interaction with human mesenchymal stromal cells.

Specific cell-cell junctions between hematopoietic stem cells (HSC) and their niche have been shown to regulate stem cell function. N-cadherin was suggested to play a central role in this process, whereas other studies indicated that it did not play an essential role in the murine model. We have analyzed the role of N-cadherin for interaction between hematopoietic progenitor cells (HPC) and supportive mesenchymal stromal cells (MSC) in a human-human setting. Expression of N-cadherin and of cadherin-11 (osteoblast cadherin) was analyzed in HPC by quantitative RT-PCR, Western blot, and flow cytometry. N-cadherin and cadherin-11 were expressed in HPC at a moderate level, whereas they were not detectable in differentiated cells. Confocal laser scanning microscopy revealed that N-cadherin and beta-catenin are colocalized at the junction of HPC and MSC. siRNA knockdown of N-cadherin or cadherin-11 as well as treatment with the blocking function antibody decreased adhesive interaction of HPC to MSC. Furthermore, knockdown of N-cadherin or blocking function antibody impaired maintenance of long-term culture-initiating cells (LTC-IC) on coculture of HPC and MSC. These results indicate that N-cadherin is involved in the bidirectional interaction of human HPC with their cellular determinants in the niche.

Aging and replicative senescence have related effects on human stem and progenitor cells.

The regenerative potential diminishes with age and this has been ascribed to functional impairments of adult stem cells. Cells in culture undergo senescence after a certain number of cell divisions whereby the cells enlarge and finally stop proliferation. This observation of replicative senescence has been extrapolated to somatic stem cells in vivo and might reflect the aging process of the whole organism. In this study we have analyzed the effect of aging on gene expression profiles of human mesenchymal stromal cells (MSC) and human hematopoietic progenitor cells (HPC). MSC were isolated from bone marrow of donors between 21 and 92 years old. 67 genes were age-induced and 60 were age-repressed. HPC were isolated from cord blood or from mobilized peripheral blood of donors between 27 and 73 years and 432 genes were age-induced and 495 were age-repressed. The overlap of age-associated differential gene expression in HPC and MSC was moderate. However, it was striking that several age-related gene expression changes in both MSC and HPC were also differentially expressed upon replicative senescence of MSC in vitro. Especially genes involved in genomic integrity and regulation of transcription were age-repressed. Although telomerase activity and telomere length varied in HPC particularly from older donors, an age-dependent decline was not significant arguing against telomere exhaustion as being causal for the aging phenotype. These studies have demonstrated that aging causes gene expression changes in human MSC and HPC that vary between the two different cell types. Changes upon aging of MSC and HPC are related to those of replicative senescence of MSC in vitro and this indicates that our stem and progenitor cells undergo a similar process also in vivo.

Replicative senescence of mesenchymal stem cells: a continuous and organized process.

Mesenchymal stem cells (MSC) comprise a promising tool for cellular therapy. These cells are usually culture expanded prior to their application. However, a precise molecular definition of MSC and the sequel of long-term in vitro culture are yet unknown. In this study, we have addressed the impact of replicative senescence on human MSC preparations. Within 43 to 77 days of cultivation (7 to 12 passages), MSC demonstrated morphological abnormalities, enlargement, attenuated expression of specific surface markers, and ultimately proliferation arrest. Adipogenic differentiation potential decreased whereas the propensity for osteogenic differentiation increased. mRNA expression profiling revealed a consistent pattern of alterations in the global gene expression signature of MSC at different passages. These changes are not restricted to later passages, but are continuously acquired with increasing passages. Genes involved in cell cycle, DNA replication and DNA repair are significantly down-regulated in late passages. Genes from chromosome 4q21 were over-represented among differentially regulated transcripts. Differential expression of 10 genes has been verified in independent donor samples as well as in MSC that were isolated under different culture conditions. Furthermore, miRNA expression profiling revealed an up-regulation of hsa-mir-371, hsa-mir-369-5P, hsa-mir-29c, hsa-mir-499 and hsa-let-7f upon in vitro propagation. Our studies indicate that replicative senescence of MSC preparations is a continuous process starting from the first passage onwards. This process includes far reaching alterations in phenotype, differentiation potential, global gene expression patterns, and miRNA profiles that need to be considered for therapeutic application of MSC preparations.

The many facets of SDF-1alpha, CXCR4 agonists and antagonists on hematopoietic progenitor cells.

Stromal cell-derived factor-1alpha (SDF-1alpha) has pleiotropic effects on hematopoietic progenitor cells (HPCs). We have monitored podia formation, migration, proliferation, and cell-cell adhesion of human HPC under the influence of SDF-1alpha, a peptide agonist of CXCR4 (CTCE-0214), a peptide antagonist (CTCE-9908), and a nonpeptide antagonist (AMD3100). Whereas SDF-1alpha induced migration of CD34(+) cells in a dose-dependent manner, CTCE-0214, CTCE-9908, and AMD3100 did not induce chemotaxis in this concentration range albeit the peptides CTCE-0214 and CTCE-9908 increased podia formation. Cell-cell adhesion of HPC to human mesenchymal stromal cells was impaired by the addition of SDF-1alpha, CTCE-0214, and AMD3100. Proliferation was not affected by SDF-1alpha or its analogs. Surface antigen detection of CXCR4 was reduced upon treatment with SDF-1alpha or AMD3100 and it was enhanced by CTCE-9908. Despite the fact that all these molecules target the same CXCR4 receptor, CXCR4 agonists and antagonists have selective effects on different functions of the natural molecule.