Adherent Cell Population Research Articles

Development of a surrogate angiogenic potency assay for clinical-grade stem cell production

Clinical results from acute myocardial infarction (AMI) patients treated with MultiStem®, a large-scale expanded adherent multipotent progenitor cell population (MAPC), have demonstrated a strong safety and benefit profile for these cells. The mechanism of benefit with MAPC treatment is a result, in part, of its ability to induce neovascularization through trophic support. Production of clinical-grade stem cell products requires the development of lot-release criteria based on potency assays that directly reflect the fundamental mechanistic pathway underlying the therapeutic response to verify manufacturing process consistency and product potency. Using an in vitro endothelial tube formation assay, a potency assay has been developed that reflects MAPC pro-angiogenic activity. Serum-free conditioned media collected from MAPC culture induced endothelial tube formation. A proteomic survey of angiogenic factors produced by the cells in vitro revealed candidate factors linked to angiogenic potency. Three cytokines, chemokine (C-X-C motif) ligand 5 (CXCL5), interleukin 8 (IL-8) and vascular endothelial growth factor (VEGF), were required for this angiogenic activity. Depletion of any of these factors from the media prevented tube formation, while adding back increasing amounts of these cytokines into the depleted serum-free conditioned media established the lower limits of each of the cytokines required to induce angiogenesis. A necessary threshold of angiogenic factor expression was established using an in vitro angiogenesis assay. By correlating the levels of the cytokines required to induce tube formation in vitro with levels of the factors found in the spent media from manufacturing production runs, detection of these factors was identified as a surrogate potency assay with defined pass/fail criteria.

Skin-derived multipotent stromal cells – an archrival for mesenchymal stem cells

Progenitor stem cells have been identified, isolated and characterized in numerous tissues and organs. However, their therapeutic potential and the use of these stem cells remain elusive except for a few progenitor cells from bone marrow, umbilical cord blood, eyes and dental pulp. The use of bone marrow-derived hematopoietic stem cells (HSC) or mesenchymal stem cells (MSCs) is restricted due to their extreme invasive procedures, low differentiation potential with age and rejection. Thus, we need a clinical grade alternative to progenitor stem cells with a high potential to differentiate, naïve and is relatively easy in in vitro propagation. In this review, we summarize cell populations of adherent and floating spheres derived from different origins of skin, or correctly foreskin, by enzymatic digestion compared with established MSCs. The morphology, phenotype, differentiation capability and immunosuppressive property of the adherent cell populations are comparable with MSCs. Serum-free cultured floating spheres have limited mesodermal but higher neurogenic differentation potential, analogous to neural crest stem cells. Both the populations confirmed their plethora potential in in vitro. Together, it may be noted that the skin-derived adherent cell populations and floating cells can be good alternative sources of progenitor cells especially in cosmetic, plastic and sports regenerative medicine.

Human Mesenchymal Stem Cells Derived From Limb Bud Can Differentiate into All Three Embryonic Germ Layers Lineages

Mesenchymal stem cells (MSCs) have been isolated from many sources, including adults and fetuses. Previous studies have demonstrated that, compared with their adult counterpart, fetal MSCs with several remarkable advantages may be a better resource for clinical applications. In this study, we successfully isolated a rapidly proliferating cell population from limb bud of aborted fetus and termed them "human limb bud-derived mesenchymal stem cells" (hLB-MSCs). Characteristics of their morphology, phenotype, cell cycle, and differentiation properties were analyzed. These adherent cell populations have a typically spindle-shaped morphology. Flow cytometry analysis showed that hLB-MSCs are positive for CD13, CD29, CD90, CD105, and CD106, but negative for CD3, CD4, CD5, CD11b, CD14, CD15, CD34, CD45, CD45RA, and HLA-DR. The detection of cell cycle from different passages indicated that hLB-MSCs have a similar potential for propagation during long culture in vitro. The most novel finding here is that, in addition to their mesodermal differentiation (osteoblasts and adipocytes), hLB-MSCs can also differentiated into extramesenchymal lineages, such as neural (ectoderm) and hepatic (endoderm) progenies. These results indicate that hLB-MSCs have a high level of plasticity and can differentiate into cell lineages from all three embryonic layers in vitro.

How are Bovine Nucleus Pulposus Cell Populations Affected by Cell Isolation Methods?

Introduction Strategies to promote intervertebral disk (IVD) regeneration have been hindered by the lack of knowledge of IVD fundamental cellular/molecular components. To date, many studies have been published culturing bovine IVD cells as a model to study IVD. However, the cellular mechanisms behind IVD de(re)generation remain largely unexplored. One of the key points to address is the characterization of the nucleus pulposus (NP) cell population(s), for which the isolation method is not consensual among the literature. These cells can be isolated using different enzymes and tissue digestion methods, but the effect of these parameters on the phenotype of cell population(s) has not been properly explored. This work aims to evaluate how the cell isolation method affects NP cell population(s). Materials and Methods NP cells were harvested from bovine caudal discs using six different digestion protocols - mechanical or enzymatic digestion, either with collagenase I, collagenase II, or both collagenase types (I + II). For mechanical tissue disruption, a gentleMACSDissociator was used. The dissociation time ranged from 4 to 19 hours. The effect of the dissociation method on NP cells immediately upon isolation was assessed by measuring cell yield (by counting cells), by viability (by flow cytometry), and by evaluating the cells morphology (by Imaging Flow Cytometry). Total 24 hours after tissue digestion, nonadherent cells were separated from the adherent fraction and the metabolic activity of both fractions was determined over time using resazurin assay. Prospective identification of different cell populations within bovine NP was performed using flow cytometry. Results Using the isolation methods described, the cell yield ranged from 4.9×105 to 1.1 × 106 cells per gram of tissue, with a cell viability between 59 and 82%. Comparing the different methods, collagenase type II digestion led to a higher cell yield (1.1 × 106 cells/g) and viability (82%). Cells isolated by the different protocols could be further characterized in terms of their morphology by measuring diverse parameters, such as cell length, elongation, shape, diameter, number of cell nucleus lobes, and number of vacuoles. Interestingly, three subpopulations of viable cells, with different sizes and auto-fluorescence, could be consistently identified by flow cytometry analysis, immediately upon isolation, without significant differences between the isolation protocols. Furthermore, two cell populations, adherent and nonadherent, could be isolated and cell metabolic activity suggests that it is possible to maintain both adherent and suspension cell populations in culture. Currently, the expression of specific cell markers for these populations is being evaluated. Conclusion Bovine NP cells were isolated using different separation methods. Digestion protocols using collagenase type II rendered higher cell yields and greater viability. Three different subpopulations of bovine NP cells were identified upon cell isolation. In monolayer culture two different cell subpopulations were separated and maintained in culture-adherent and nonadherent NP cells. Overall, this study demonstrates that the isolation method is crucial for the subsequent study of bovine NP cells. The identification of different NP cell populations and their dependence on the isolation method is particularly important when envisioning the identification of novel biological evidences that contribute to highlight IVD degeneration and its potential regenerative capacity. Acknowledgments This work was financed by FEDER funds through the Programa Operacional Factores de Competitividade - COMPETE, by portuguese funds through FCT - Fundação para a Ciência e a Tecnologia. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared

Hematopoietic progenitor constituents and adherent cell progenitor morphology isolated from black‐rumped agouti (Dasyprocta prymnolopha, Wagler 1831) bone marrow

Stem cells are present in the adult tissues of most diverse species. Bone marrow is recognized to be the most exploited site to obtain stem cells and cell progenitors. The objective of the present study was to characterize hematopoietic progenitor (HP) morphology and analyze the performance of adherent cell progenitors (ACPs) cultivated in vitro from black-rumped agouti bone marrow (Dasyprocta prymnolopha). Bone marrow aspirates were obtained from tibia crest and used to prepare histological slides and identify cell morphology. Cells were also scattered on culture plates for later isolation, expansion, and quantification. Smears obtained from bone marrow demonstrated HPs at different stages of maturity. In culture, these cells showed fibroblastoid morphology and a strong tendency to form colonies, demonstrated by the presence of cell aggregates, cytoplasmic elongations lying side by side. An 80% cell confluence was observed at 18 days in culture and progressive reduction in the percentage of nonadherent mononuclear cells. After eight passes, a mean cell viability of 96.07% was observed, from a pool of 1.6 × 10(7) cells (ACP). Thirteen 25-cm(2) culture bottles were trypsinized, resuspended in freezing medium, stored in 14 criotubes at a concentration of 1 × 10(6) cells per milliliter, and placed in liquid nitrogen at -196°C. Agouti bone marrow demonstrated high plasticity, moreover different HP lines, and a population of adherent cells demonstrated morphology similar to mesenchymal stem cells in culture.

Clinical Applications and Biosafety of Human Adult Mesenchymal Stem Cells

Mesenchymal Stem Cells (MSCs) are a population of adherent cells that can differentiate into mesenchymal lineage populations (cartilage, bone and fat tissue). In addition, they seem to be able to differentiate also into a broader type of lineages other than the original mesodermal germ layer. Bone marrow MSCs are a standard in the field of adult stem cell biology and clinical applications; however adipose-derived MSCs are becoming an attractive alternative due to their minimally invasive accessibility and availability in the body. MSCs modulate several effector immune functions by interacting both with innate and adoptive immune responses. Several local signals from the tissue microenvironment, together with cytokine and soluble factors released by MSCs influence anti-inflammatory and tissue repair properties of infused MSCs. Therefore, cellular therapies utilizing ex vivo expanded MSCs may be an interesting approach for inflammatory and autoimmune diseases. Biosafety is still one of the most important aspects; therefore the production of clinical-grade MSCs requires the careful identification and control of all the phases of cell manipulation and release. Many clinical applications of adult MSCs are in progress and are using bone marrow or adipose tissue-derived MSCs for the treatment of Graft Versus Host Disease (GVHD), inflammatory joint diseases and osteocartilagineous defects, digestive tract, cardiovascular and neurological diseases.

Abstract 1478: Bone marrow mesenchymal stem cells suppress tumor cell apoptosis and are selectively recruited to early-stage tumors

Abstract Bone marrow mesenchymal stem cells (BMMSC) are recruited to primary tumors and have been reported to display pro-tumorigenic as well as anti-tumorigenic activities. However, the mechanisms behind their effect on tumor development are still unclear. We have isolated murine bone marrow aspirates and plated them on fibronectin/collagen I, which allows expansion of an adherent stromal cell population in 3-6 weeks. These cells express the CD44 hyaluronic acid receptor in the absence of hematopoietic (CD34), myeloid (CD45), and endothelial (CD31/VEGFR2) markers and display both spontaneous and inducible adipogenesis/ osteoblastogenesis in culture. 3D matrigel cultures of murine 4T1 mammary carcinoma cells revealed enhanced generation of tumor spheroids in the presence of BMMSC or their conditioned media. Conditioned media from BMMSC also increased 4T1 cell expansion in 2D monolayer cultures, with loss of this phenomenon after heat inactivation suggesting the presence of protein-based signaling. Cell cycle analysis of 4T1 cells treated with BMMSC conditioned media revealed no statistically significant change in mitotic potential in either growth (serum present) or growth-arrest (serum depleted) phase, as determined by PI staining and BrdU uptake. However, analysis of apoptosis in 4T1 cells treated with BMMSC conditioned media showed increased percentages of live cells by PI uptake and AnnexinV staining, as well as a reduction in caspase-3 and caspase-9 activation by colorimetric assay, as the cells entered growth arrest phase. Furthermore, treatment with BMMSC conditioned media preserved viability in 4T1 cells exposed to paclitaxel under no-serum conditions. Complementary studies of BMMSC tumor-directed migration indicate that BMMSC show preferential in vitro recruitment by murine tumor cell lines which generate reactive stroma (4T1, LL/2 lung carcinoma) vs. no stroma (B16 melanoma). In vivo recruitment of BMMSC by early-stage/slow-growing subcutaneous 4T1 tumors was also visualized by bioluminescent imaging, red fluorescence, and magnetic resonance imaging of BMMSC labeled with iron-nanoparticles. These results were confirmed by immunohistochemical evaluation of iron deposits by Prussian blue stain and by immunofluorescent detection of PKH26-labeled BMMSC. Overall, our data identify BMMSC as an important mediator of tumor cell survival/treatment resistance and reveal an enhanced tropism of BMMSC toward early 4T1 tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1478. doi:1538-7445.AM2012-1478

Microscale functional cytomics for studying hematologic cancers

AbstractAn important problem in translational cancer research is our limited ability to functionally characterize behaviors of primary patient cancer cells and associated stromal cell types, and relate mechanistic understanding to therapy selection. Functional analyses of primary samples face at least 3 major challenges: limited availability of primary samples for testing, paucity of functional information extracted from samples, and lack of functional methods accessible to many researchers. We developed a microscale cell culture platform that overcomes these limitations, especially for hematologic cancers. A key feature of the platform is the ability to compartmentalize small populations of adherent and nonadherent cells in controlled microenvironments that can better reflect physiological conditions and enable cell-cell interaction studies. Custom image analysis was developed to measure cell viability and protein subcellular localizations in single cells to provide insights into heterogeneity of cellular responses. We validated our platform by assessing viability and nuclear translocations of NF-κB and STAT3 in multiple myeloma cells exposed to different conditions, including cocultured bone marrow stromal cells. We further assessed its utility by analyzing NF-κB activation in a primary chronic lymphocytic leukemia patient sample. Our platform can be applied to myriad biological questions, enabling high-content functional cytomics of primary hematologic malignancies.

Improved Culture-Based Isolation of Differentiating Endothelial Progenitor Cells from Mouse Bone Marrow Mononuclear Cells

Numerous endothelial progenitor cell (EPC)-related investigations have been performed in mouse experiments. However, defined characteristics of mouse cultured EPC have not been examined. We focused on fast versus slow adherent cell population in bone marrow mononuclear cells (BMMNCs) in culture and examined their characteristics. After 24 h-culture of BMMNCs, attached (AT) cells and floating (FL) cells were further cultured in endothelial differentiation medium separately. Immunological and molecular analyses exhibited more endothelial-like and less monocyte/macrophage-like characteristics in FL cells compared with AT cells. FL cells formed thick/stable tube and hypoxia or shear stress overload further enhanced these endothelial-like features with increased angiogenic cytokine/growth factor mRNA expressions. Finally, FL cells exhibited therapeutic potential in a mouse myocardial infarction model showing the specific local recruitment to ischemic border zone and tissue preservation. These findings suggest that slow adherent (FL) but not fast attached (AT) BMMNCs in culture are EPC-rich population in mouse.

Evaluation of a Thin and Mechanically Stable Collagen Cell Carrier

The biological function of adherent cell populations strongly depends on the physical and biochemical properties of extracellular matrix molecules. Therefore, numerous biocompatible cell carriers have been developed to specifically influence cell attachment, proliferation, cellular differentiation, and tissue formation for diverse cell culture applications and cell-based therapies. In the present study, we evaluated the mechanical and the cell biological properties of a novel, thin, and planar collagen scaffold. The cell carrier is based on fibrillar bovine collagen type I and exhibits a low material thickness coupled with a high mechanical stability as measured by tensile tests. The influence of this new biomaterial on cell viability, proliferation, and cell differentiation was analyzed using 5-bromo-2-deoxyuridine (BrdU) proliferation assay, immunocytochemistry, water-soluble tetrazolium salt-1 assay (WST-1), live cell imaging, and electron microscopy. Cell culture experiments with the human osteosarcoma cell line Saos-2, human mesenchymal stem cells, and rodent cardiomyocytes demonstrated the in vitro biocompatibility of this chemically noncrosslinked scaffold. Both the mechanical characteristics and the in vitro biocompatibility of this collagen type I carrier facilitate the engineering of thin transferable tissue constructs and offer new possibilities in the fields of cell culture techniques, tissue engineering, and regenerative medicine.

Safety and feasibility of third-party multipotent adult progenitor cells for immunomodulation therapy after liver transplantation--a phase I study (MISOT-I)

BackgroundLiver transplantation is the definitive treatment for many end-stage liver diseases. However, the life-long immunosuppression needed to prevent graft rejection causes clinically significant side effects. Cellular immunomodulatory therapies may allow the dose of immunosuppressive drugs to be reduced. In the current protocol, we propose to complement immunosuppressive pharmacotherapy with third-party multipotent adult progenitor cells (MAPCs), a culture-selected population of adult adherent stem cells derived from bone marrow that has been shown to display potent immunomodulatory and regenerative properties. In animal models, MAPCs reduce the need for pharmacological immunosuppression after experimental solid organ transplantation and regenerate damaged organs.MethodsPatients enrolled in this phase I, single-arm, single-center safety and feasibility study (n = 3-24) will receive 2 doses of third-party MAPCs after liver transplantation, on days 1 and 3, in addition to a calcineurin-inhibitor-free "bottom-up" immunosuppressive regimen with basiliximab, mycophenolic acid, and steroids. The study objective is to evaluate the safety and clinical feasibility of MAPC administration in this patient cohort. The primary endpoint of the study is safety, assessed by standardized dose-limiting toxicity events. One secondary endpoint is the time until first biopsy-proven acute rejection, in order to collect first evidence of efficacy. Dose escalation (150, 300, 450, and 600 million MAPCs) will be done according to a 3 + 3 classical escalation design (4 groups of 3-6 patients each).DiscussionIf MAPCs are safe for patients undergoing liver transplantation in this study, a phase II/III trial will be conducted to assess their clinical efficacy.

Analysis of a simulated in vivo environment for selective induction of apoptosis

Apoptosis is thought to play a major role in biological processes and in establishing effective treatments for cancer. The objective of this study was to examine the response of human erythroleukemia (HEL) cells when exposed to known apoptosis inducers and to analyze a simulated in vivo environment to determine whether normal cells (WI‐38 cells) and HEL cells were affected by the chemicals.The HEL cells were treated with camptothecin (CP), vehicle alone, and various heteroarotinoids (HETS) twenty‐four hr after subculture and incubated for an additional 48–72 hr. The cells were harvested and monitored for apoptosis, growth, and viability. Apoptosis was quantitated using the Hoechst/propidium iodide assay. Preliminary results proved one of the five HETS tested had significant elevated levels of apoptosis when compared to the vehicle alone.In an attempt to mimic an in vivo environment, HEL cells, grown in suspension, were introduced to a normal human adherent cell population at various cell densities and allowed to incubate for 72 hr. The majority of the HEL cells adhered to the WI‐38 cells. A large population of HEL cells was resistant to removal from the WI‐38 cells following a vigorous shake, suggesting a strong bond between the cells. In order to determine the selectivity of apoptosis, CP was added to the system. The initial results suggest that both cell types were negatively affected by CP. Support was provided by SRU.

Nitric oxide changes distinct aspects of the glycophenotype of human neuroblastoma NB69 cells

It is an open question whether the presence of nitric oxide (NO) affects the cell glycophenotype. A panel of six plant lectins was used in this study to monitor distinct aspects of cell surface glycosylation under nitrosative stress. We determined that treating human neuroblastoma NB69 cells with the long-lived NO donor 2,2′-(hydroxynitrosohydrazono)bis-ethanimine (DETA/NO) and monitoring the non-apoptotic adherent cell population significantly increases the presentation of N-glycans as detected by concanavalin A. Examining fine-structural features, bisected N-glycans and branch-end tailoring including α2,6-sialylation were found to be enhanced. Confocal fluorescence microscopy and cell permeabilization experiments pointed to a major effect of NO on the extent of cell surface N-glycan presentation. We also show that NO increases the level of protein O-GlcNAcylation, a multifunctional post-translational modification. Our results thus establish the first evidence for NO as modulator of distinct aspects of cell glycosylation.

DLK-1 as a Marker to Distinguish Unrestricted Somatic Stem Cells and Mesenchymal Stromal Cells in Cord Blood

In addition to hematopoietic stem cells, cord blood (CB) also contains different nonhematopoietic CD45-, CD34- adherent cell populations: cord blood mesenchymal stromal cells (CB MSC) that behave almost like MSC from bone marrow (BM MSC) and unrestricted somatic stem cells (USSC) that differentiate into cells of all 3 germ layers. Distinguishing between these populations is difficult due to overlapping features such as the immunophenotype or the osteogenic and chondrogenic differentiation pathway. Functional differences in the differentiation potential suggest different developmental stages or different cell populations. Here we demonstrate that the expression of genes and the differentiation toward the adipogenic lineage can discriminate between these 2 populations. USSC, including clonal-derived cells lacking adipogenic differentiation, strongly expressed δ-like 1/preadipocyte factor 1 (DLK-1/PREF1) correlating with high proliferative potential, while CB MSC were characterized by a strong differentiation toward adipocytes correlating with a weak or negative DLK-1/PREF1 expression. Constitutive overexpression of DLK-1/PREF1 in CB MSC resulted in a reduced adipogenic differentiation, whereas silencing of DLK-1 in USSC resulted in adipogenic differentiation.

Effective enrichment of prostate cancer stem cells from spheres in a suspension culture system

BackgroundStem-like prostate cancer cells are also called prostate cancer stem cells (PrCSCs). These rare cells are supposed to be highly tumorigenic and to be involved in maintenance of tumor homeostasis and mediation of tumor metastasis. Methods for sorting PrCSCs are mainly based on sorting cells with the marker (CD133+/CD44+) or side population cells. However, CD133+/CD44+ cells or side population cells are very rare or even undetectable. The scarcity of approaches for isolation and purification of PrCSCs is the main obstacle to studying PrCSCs. MethodsIn the present study, suspension culture was used for enrichment of PrCSCs. And PrCSCs were verified by side population technology, drug sensitivity assays, and the molecular marker analysis of prostate cancer stem cell. ResultsPC3 cells survived and formed spheres in nonadherent suspension culture. The percentage of CD44+/CD133+ cells was 18-fold higher in the nonadherent sphere-forming cell population than in the adherent PC3 cell population (13.94% vs. 0.77%, respectively). This side population was increased to 3.1% in the nonadherent population but undetectable in adherent population. Resistance to cisplatin was higher in the nonadherent cells than adherent cells. ConclusionSuspension culture can be used to enrich for PrCSCs. This approach will aid prostate stem cell biology research and facilitate identification of novel therapeutic agents for prostate cancer.

Isolation and characterization of true mesenchymal stem cells derived from human term decidua capable of multilineage differentiation into all 3 embryonic layers

The objective of the study was to isolate and characterize a population of mesenchymal stem cells (MSCs) from human term placental membranes. We isolated an adherent cell population from extraembryonic membranes. Morphology, phenotype, growth characteristics, karyotype, and immunological and differentiation properties were analyzed. The isolated placental MSCs were from maternal origin and named as decidua-derived mesenchymal stem cells (DMSCs). DMSCs differentiated into derivatives of all germ layers. It is the first report about placental MSC differentiation into alveolar type II cells. Clonally expanded DMSCs differentiated into all embryonic layers, including pulmonary cells. DMSCs showed higher life span than placental cells from fetal origin and proliferated without genomic instability. The data suggest that DMSCs are true multipotent MSCs, distinguishing them from other placental MSCs. DMSCs could be safely used in the mother as a potential source of MSCs for pelvic floor dysfunctions and immunological diseases. Additionally, frozen DMSCs can be stored for both autologous and allogeneic tissue regeneration.

Characterization of Adipose-Derived Stem Cells: An Update

Adipose tissue is an attractive source of multipotent adult stem cells due to its wide-spread availability, accessibility, and ease of harvest. Adipose-derived stem cells (ASCs), the adherent stromal cell population present within adipose tissue, are easily expanded in culture, able to differentiate along multiple cell-lineage pathways, and have been shown to provide therapeutic benefit in models of injury and disease through immunomodulation, structural integation, and/or trophic support. Recent developments in the characterization of ASCs, specifically their isolation, gene and protein expression, differentiation, and expansion, are reviewed in this article.

Quantification of Fluorescence Intensity of Labeled Human Mesenchymal Stem Cells and Cell Counting of Unlabeled Cells in Phase-Contrast Imaging: An Open-Source-Based Algorithm

Assessment of cell fate is indispensable to evaluate cell-based therapies in regenerative medicine. Therefore, a widely used technique is fluorescence labeling. A major problem still is the standardized, noninvasive, and reliable quantification of fluorescence intensity of adherent cell populations on single-cell level, since total fluorescence intensity must be correlated to the cell number. Consequently, the aim of the present study was to produce and validate an open-source-based algorithm, capable of measuring the total fluorescence intensity of cell populations and assessing the total cell number in phase-contrast images. To verify the algorithms' capacity to assess fluorescence intensity, human mesenchymal stem cells were transduced to stably express enhanced green fluorescent protein and results produced by the algorithm were compared to flow cytometry analysis. No significant differences could be observed at any time (p ≥ 0.443). For validation of the algorithm for cell counting in phase-contrast images, adherent human mesenchymal stem cells were manually counted and compared to results produced by the algorithm (correlation coefficient [CC] r = 0.975), nuclei staining (CC r = 0.997), and hemocytometer (CC r = 0.629). We conclude that applying the developed algorithm in routine practice allows robust, fast, and reproducible assessment of fluorescence intensity and cell numbers in simple large-scale microscopy. The method is easy to perform and open source based.

The HOX Code as a “biological fingerprint” to distinguish functionally distinct stem cell populations derived from cord blood

Mesenchymal stem cells (MSC) have been isolated from almost every adult tissue. In cord blood (CB), different non-hematopoietic CD45-, CD34− adherent cell populations can be generated: the cord blood derived MSC (CB-MSC), that behave almost like MSC from bone marrow (BM-MSC), and unrestricted somatic stem cells (USSC) which show a distinct differentiation potential into all three germ layers. However, distinguishing these populations easily by molecular markers is still a concern. In this study we were able to present the HOX expression pattern of USSC, CB-MSC and BM-MSC, which in fact allows a discrimination of these populations. Briefly, RT-PCR analysis of the HOX code revealed a high similarity between BM-MSC and CB-MSC, which are both HOX-positive, whereas USSC resembled H9 embryonic stem cells HOX-negative.Especially HOXA9, HOXB7, HOXC10 and HOXD8 are good candidate markers to discriminate MSC from USSC. Thus, our data suggest that the "biological fingerprint" based on the HOX code can be used to distinguish functionally distinct MSC populations derived from bone marrow and cord blood.

Continuous separation of cells of high osteoblastic differentiation potential from mesenchymal stem cells on an antibody-immobilized column

Here, we report that two distinctive cell populations with osteoblastic differentiation ability were found in adherent cell populations from bone marrow. Mesenchymal stem cells (MSCs) were conventionally isolated by using adherent property of bone marrow cells onto a plastic culture dish. MSCs enriched on the basis of their adherent property were considered phenotypically and functionally heterogeneous. We developed a ligand-immobilized surface for separating subpopulation of adherent cells derived from bone marrow by the cell rolling process. We successfully isolate two cell populations with high differentiation ability for osteoblasts in adherent bone marrow cells by using the anti-CD34 antibody-immobilized column. The antibody was covalently conjugated with polyacrylic acid and introduced onto the inner surface of a silicone tube. When cell suspension of MSCs was injected into the antibody-immobilized column, different cell populations were isolated. After the cultivation of isolated cells in the osteoblastic differentiation medium for 1 week, few sub-populations were strongly induced to form osteoblastic cells. This study revealed that the ligand-immobilized surface can be used to continually separate cell populations under a labeling-free condition.