Effect of geometrical constraints on human pluripotent stem cell nuclei in pluripotency and differentiation.

Mechanical stimuli and geometrical constraints transmitted across the cytoskeleton to the nucleus affect the nuclear morphology and cell function. Human pluripotent stem cells (hPSCs) represent an effective tool for evaluating transitions in nuclear deformability from the pluripotent to differentiated stage, and for deciphering the underlying mechanisms. We report the first study that investigates the nuclear deformability induced by geometrical constraints of hPSCs both in the pluripotent stage and during early germ layer specification. We specifically developed micro-structured surfaces coupled with high-content imaging analysis algorithms to quantitatively characterize nuclear deformability. Our results show that hPSCs possess high nuclear deformability, which does not alter pluripotency. We observed nuclear deformability transition along early germ layer specification: during early ectoderm differentiation nuclear deformability is strongly reduced, during early endoderm differentiation nuclei keep a deformed shape and during early mesoderm specification they show an intermediate behaviour. Different mRNA expressions between hPSCs differentiated on flat and micro-structured surfaces have been observed along early mesoderm and early endoderm specification. In order to better understand the mechanisms of the nuclear deformability transition observed during early ectoderm differentiation, we also employed cytoskeletal and nuclear protein inhibitors to evaluate their role in determining the nuclear shape. Actin and nesprin are essential for maintaining deformed nuclei, while lamin A/C and intermediate filaments confer rigidity to the nucleus. This study suggests that nuclear deformability is highly regulated during differentiation.

Collagen and Hydroxyapatite Scaffolds Activate Distinct Osteogenesis Signaling Pathways in Adult Adipose-Derived Multipotent Stromal Cells.

Osteogenic cell signaling pathway disruption varies among bone diseases. This investigation was designed to identify adipose-derived multipotent stromal cell (ASC) and bone graft scaffold combinations for local, targeted restoration of gene expression and extracellular matrix (ECM) deposition. Human ASC osteogenesis on bone graft materials was quantified following culture in stromal (S), osteogenic (O), or osteogenic for 48 h followed by stromal medium (OS) to test the two-part hypothesis: (1) identical ASC isolates on distinct bone graft scaffolds demonstrate unique viability, differentiation, ECM production, and gene expression in the same culture conditions; (2) identical ASC-bone graft scaffold combinations have different cell viability, differentiation, ECM production, and gene expression when cultured in S, O, or OS medium. Three commercially available bone graft scaffold materials, type I bovine collagen (C), hydroxyapatite + ß-tricalcium phosphate + type I bovine collagen (HT), and ß-tricalcium phosphate + type I bovine collagen (CT) were evaluated. Passage 3 ASCs were loaded onto scaffold blocks with a spinner flask bioreactor, and constructs were cultured up to 28 days. Cell viability, gene expression (alkaline phosphatase [ALPL], osteoprotegerin [TNFRSF11B], osteocalcin [BGLAP], cannabinoid receptors type I [CNR1] and II [CNR2], receptor activator of nuclear factor kappa ß ligand [TNFSF11]), as well as ECM DNA, collagen, sulfated glycosaminoglycan, and protein content were quantified. Matrix organization was evaluated with scanning electron microscopy. Effects of scaffold, medium, or culture duration on cell viability were minimal. Significantly higher initial ALPL expression decreased with time, while BGLAP expression increased in HT constructs in O medium, and the constructs had the most abundant ECM components and ultrastructural organization. There was a similar, although delayed, pattern of gene expression and greater ECM collagen with less organization in C constructs in O medium. Higher CNR1 expression in C versus higher TNFRSF11B/TNFSF11 expression in HT constructs throughout the study support stimulation of unique osteogenic signaling pathways by identical cell isolates. These results suggest that bone scaffold composition may be used to selectively target specific osteogenic cell signaling pathways in ASC constructs to stimulate ECM deposition based on therapeutic needs.

Cells with surface expression of CD133highCD71low are enriched for tripotent colony-forming progenitor cells in the adult murine pancreas.

Progenitor cells in the adult pancreas are potential sources of endocrine beta cells for treating type 1 diabetes. Previously, we identified tri-potent progenitor cells in the adult (2-4month-old) murine pancreas that were capable of self-renewal and differentiation into duct, acinar, and endocrine cells in vitro. These progenitor cells were named pancreatic colony-forming units (PCFUs). However, because PCFUs are a minor population in the pancreas (~1%) they are difficult to study. To enrich PCFUs, strategies using cell-surface marker analyses and fluorescence-activated cell sorting were developed. We found that CD133(high)CD71(low) cells, but not other cell populations, enriched PCFUs by up to 30 fold compared to the unsorted cells. CD133(high)CD71(low) cells generated primary, secondary, and subsequent colonies when serially re-plated in Matrigel-containing cultures, suggesting self-renewal abilities. In the presence of a laminin hydrogel, CD133(high)CD71(low) cells gave rise to colonies that contained duct, acinar, and Insulin(+)Glucagon(+) double-hormonal endocrine cells. Colonies from the laminin hydrogel culture were implanted into diabetic mice, and five weeks later duct, acinar, and Insulin(+)Glucagon(-) cells were detected in the grafts, demonstrating tri-lineage differentiation potential of CD133(high)CD71(low) cells. These CD133(high)CD71(low) cells will enable future studies of putative adult pancreas stem cells in vivo.

The effect of the bioactive sphingolipids S1P and C1P on multipotent stromal cells--new opportunities in regenerative medicine.

Sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) belong to a family of bioactive sphingolipids that act as important extracellular signaling molecules and chemoattractants. This study investigated the influence of S1P and C1P on the morphology, proliferation activity and osteogenic properties of rat multipotent stromal cells derived from bone marrow (BMSCs) and subcutaneous adipose tissue (ASCs). We show that S1P and C1P can influence mesenchymal stem cells (MSCs), each in a different manner. S1P stimulation promoted the formation of cellular aggregates of BMSCs and ASCs, while C1P had an effect on the regular growth pattern and expanded intercellular connections, thereby increasing the proliferative activity. Although osteogenic differentiation of MSCs was enhanced by the addition of S1P, the effectiveness of osteoblast differentiation was more evident in BMSCs, particularly when biochemical and molecular marker levels were considered. The results of the functional osteogenic differentiation assay, which includes an evaluation of the efficiency of extracellular matrix mineralization (SEM-EDX), revealed the formation of numerous mineral aggregates in BMSC cultures stimulated with S1P. Our data demonstrated that in an appropriate combination, the bioactive sphingolipids S1P and C1P may find wide application in regenerative medicine, particularly in bone regeneration with the use of MSCs.

Histone methylation codes involved in stemness, multipotency, and senescence in budding tunicates.

We examined the dynamics of nuclear histone H3 trimethylation related to cell differentiation and aging in a budding tunicate, Polyandrocarpa misakiensis. Throughout zooidal life, multipotent epithelial and coelomic cell nuclei showed strong trimethylation signals at H3 lysine27 (H3K27me3), consistent with the results of western blotting. Epidermal H3K27me3 repeatedly appeared in protruding buds and disappeared in senescent adult zooids. The budding-specific cytostatic factor TC14-3 allowed aging epidermal cells to restore H3K27me3 signals and mitochondrial gene activities via mitochondrial transcription factor a, all of which were made ineffective by an H3K27me3 inhibitor. Chromatin immunoprecipitation showed that TC14-3 enhances H3K27me3 of transdifferentiation-related genes and consequently downregulates the expression of these genes. In contrast, trimethylation signals at H3 lysine4 (H3K4me3) appeared transiently in transdifferentiating bud cells and stably lasted in undifferentiated adult cells without affecting H3K27me3. A transdifferentiation-related gene external signal-regulated kinase heavily underwent H3K4me3 in developing buds, which could be reproduced by retinoic acid. These results indicate that in P. misakiensis, TC14-3-driven H3K27 trimethylation is a default state of bud and zooid cells, which serves as the histone code for cell longevity. H3K27me3 and H3K4me3 double-positive signals are involved in cell stemness, and absence of signals is the indication of senescence.

Colony-forming progenitor cells in the postnatal mouse liver and pancreas give rise to morphologically distinct insulin-expressing colonies in 3D cultures.

In our previous studies, colony-forming progenitor cells isolated from murine embryonic stem cell-derived cultures were differentiated into morphologically distinct insulin-expressing colonies. These colonies were small and not light-reflective when observed by phase-contrast microscopy (therefore termed "Dark" colonies). A single progenitor cell capable of giving rise to a Dark colony was termed a Dark colony-forming unit (CFU-Dark). The goal of the current study was to test whether endogenous pancreas, and its developmentally related liver, harbored CFU-Dark. Here we show that dissociated single cells from liver and pancreas of one-week-old mice give rise to Dark colonies in methylcellulose-based semisolid culture media containing either Matrigel or laminin hydrogel (an artificial extracellular matrix protein). CFU-Dark comprise approximately 0.1% and 0.03% of the postnatal hepatic and pancreatic cells, respectively. Adult liver also contains CFU-Dark, but at a much lower frequency (~0.003%). Microfluidic qRT-PCR, immunostaining, and electron microscopy analyses of individually handpicked colonies reveal the expression of insulin in many, but not all, Dark colonies. Most pancreatic insulin-positive Dark colonies also express glucagon, whereas liver colonies do not. Liver CFU-Dark require Matrigel, but not laminin hydrogel, to become insulin-positive. In contrast, laminin hydrogel is sufficient to support the development of pancreatic Dark colonies that express insulin. Postnatal liver CFU-Dark display a cell surface marker CD133⁺CD49f(low)CD107b(low) phenotype, while pancreatic CFU-Dark are CD133⁻. Together, these results demonstrate that specific progenitor cells in the postnatal liver and pancreas are capable of developing into insulin-expressing colonies, but they differ in frequency, marker expression, and matrix protein requirements for growth.

Preferential and comprehensive reconstitution of severely damaged sciatic nerve using murine skeletal muscle-derived multipotent stem cells.

Loss of vital functions in the somatic motor and sensory nervous systems can be induced by severe peripheral nerve transection with a long gap following trauma. In such cases, autologous nerve grafts have been used as the gold standard, with the expectation of activation and proliferation of graft-concomitant Schwann cells associated with their paracrine effects. However, there are a limited number of suitable sites available for harvesting of nerve autografts due to the unavoidable sacrifice of other healthy functions. To overcome this problem, the potential of skeletal muscle-derived multipotent stem cells (Sk-MSCs) was examined as a novel alternative cell source for peripheral nerve regeneration. Cultured/expanded Sk-MSCs were injected into severely crushed sciatic nerve corresponding to serious neurotmesis. After 4 weeks, engrafted Sk-MSCs preferentially differentiated into not only Schwann cells, but also perineurial/endoneurial cells, and formed myelin sheath and perineurium/endoneurium, encircling the regenerated axons. Increased vascular formation was also observed, leading to a favorable blood supply and waste product excretion. In addition, engrafted cells expressed key neurotrophic and nerve/vascular growth factor mRNAs; thus, endocrine/paracrine effects for the donor/recipient cells were also expected. Interestingly, skeletal myogenic capacity of expanded Sk-MSCs was clearly diminished in peripheral nerve niche. The same differentiation and tissue reconstitution capacity of Sk-MSCs was sufficiently exerted in the long nerve gap bridging the acellular conduit, which facilitated nerve regeneration/reconnection. These effects represent favorable functional recovery in Sk-MSC-treated mice, as demonstrated by good corduroy walking. We also demonstrated that these differentiation characteristics of the Sk-MSCs were comparable to native peripheral nerve-derived cells, whereas the therapeutic capacities were largely superior in Sk-MSCs. Therefore, Sk-MSCs can be a novel/suitable alternative cell source for healthy nerve autografts.

Ultrastructural features of human adipose-derived multipotent mesenchymal stromal cells.

Multipotent mesenchymal stromal cells (MMSCs) are plastic-adherent cells with a well-established phenotype. Equine, but not human, adipose MMSCs have been characterized ultrastructurally. The purpose of our study was to evaluate ultrastructurally the adipose-derived human MMSCs. Cell cultures were prepared from human lipoaspirate. The flow cytometry evaluation of surface markers of cultured cells confirmed the expected profile of MMSCs, that were positive for CD73, CD90 and CD105, and negative for CD34 and CD45. We examined these human adipose-derived MMSCs in transmission electron microscopy (TEM) by Epon en-face embedding the fixed MMSCs. The main ultrastructural features of MMSCs were the extremely rich content of endosomal/vesicular elements, long mitochondria, dilated RER (rough endoplasmic reticulum) cisternae, and abundant intermediate filaments and microtubules. We found two types of MMSCS prolongations: (a) thick processes, with opposite, vesicular and filaments-rich, sides and (b) slender processes (pseudopodes and filopodes), with occasional proximal dilated segments housing mitochondria, vesicles and secretory granules. These TEM features of MMSCs characterized an in vitro cell population and could use to distinguish between different cell types in culture.

In vitro mesenchymal trilineage differentiation and extracellular matrix production by adipose and bone marrow derived adult equine multipotent stromal cells on a collagen scaffold.

Directed differentiation of adult multipotent stromal cells (MSC) is critical for effective treatment strategies. This study was designed to evaluate the capability of equine MSC from bone marrow (BMSC) and adipose tissue (ASC) on a type I collagen (COLI) scaffold to undergo chondrogenic, osteogenic and adipogenic differentiation and form extracellular matrix (ECM) in vitro. Following determination of surface antigen expression, MSC were loaded into scaffolds in a perfusion bioreactor and loading efficiency was quantified. Cell-scaffold constructs were assessed after loading and 7, 14 and 21 days of culture in stromal or induction medium. Cell number was determined with DNA content, cell viability and spatial uniformity with confocal laser microscopy and cell phenotype and matrix production with light and scanning electron microscopy and mRNA levels. The MSC were positive for CD29 (>90 %), CD44 (>99 %), and CD105 (>60 %). Loading efficiencies were >70 %. The ASC and BMSC cell numbers on scaffolds were affected by culture in induction medium differently. Viable cells remained uniformly distributed in scaffolds for up to 21 days and could be directed to differentiate or to maintain an MSC phenotype. Micro- and ultrastructure showed lineage-specific cell and ECM changes. Lineage-specific mRNA levels differed between ASC and BMSC with induction and changed with time. Based on these results, equine ASC and BMSC differentiate into chondrogenic, osteogenic and adipogenic lineages and form ECM similarly on COLI scaffolds. The collected data supports the potential for equine MSC-COLI constructs to support diverse equine tissue formation for controlled biological studies.

18F-FDG labeling of mesenchymal stem cells and multipotent adult progenitor cells for PET imaging: effects on ultrastructure and differentiation capacity.

UNLABELLED: Because of their extended differentiation capacity, stem cells have gained great interest in the field of regenerative medicine. For the development of therapeutic strategies, more knowledge on the in vivo fate of these cells has to be acquired. Therefore, stem cells can be labeled with radioactive tracer molecules such as (18)F-FDG, a positron-emitting glucose analog that is taken up and metabolically trapped by the cells. The aim of this study was to optimize the radioactive labeling of mesenchymal stem cells (MSCs) and multipotent adult progenitor cells (MAPCs) in vitro with (18)F-FDG and to investigate the potential radiotoxic effects of this labeling procedure with a range of techniques, including transmission electron microscopy (TEM). METHODS: Mouse MSCs and rat MAPCs were used for (18)F-FDG uptake kinetics and tracer retention studies. Cell metabolic activity, proliferation, differentiation and ultrastructural changes after labeling were evaluated using an Alamar Blue reagent, doubling time calculations and quantitative TEM, respectively. Additionally, mice were injected with MSCs and MAPCs prelabeled with (18)F-FDG, and stem cell biodistribution was investigated using small-animal PET. RESULTS: The optimal incubation period for (18)F-FDG uptake was 60 min. Significant early tracer washout was observed, with approximately 30%-40% of the tracer being retained inside the cells 3 h after labeling. Cell viability, proliferation, and differentiation capacity were not severely affected by (18)F-FDG labeling. No major changes at the ultrastructural level, considering mitochondrial length, lysosome size, the number of lysosomes, the number of vacuoles, and the average rough endoplasmic reticulum width, were observed with TEM. Small-animal PET experiments with radiolabeled MAPCs and MSCs injected intravenously in mice showed a predominant accumulation in the lungs and a substantial elution of (18)F-FDG from the cells. CONCLUSION: MSCs and MAPCs can be successfully labeled with (18)F-FDG for molecular imaging purposes. The main cellular properties are not rigorously affected. TEM confirmed that the cells' ultrastructural properties are not influenced by (18)F-FDG labeling. Small-animal PET studies confirmed the intracellular location of the tracer and the possibility of imaging injected prelabeled stem cell types in vivo. Therefore, direct labeling of MSCs and MAPCs with (18)F-FDG is a suitable technique to noninvasively assess cell delivery and early retention with PET.

Immunocytochemical and structural comparative study of committed versus multipotent stem cells cultured with different biomaterials.

The aim of this work was the comparison of the behavior of committed (human osteoblast cells - hOB - from bone biopsies) versus multipotent (human dental pulp stem cells - hDPSC - from extracted teeth) cells, cultured on shot-peened titanium surfaces, since the kind of cell model considered has been shown to be relevant in techniques widely used in studies on composition/morphology of biomaterial surfaces. The titanium surface morphology, with different roughness, and the behavior of cells were analyzed by confocal microscope (CM), scanning electron microscope (SEM) and X-ray microanalysis. The best results, in terms of hOB adhesion/distribution, were highlighted by both CM and SEM in cultured plates having 20-µm-depth cavities. On the contrary, CM and SEM results highlighted the hDPSC growth regardless the different surface morphology, arranged in overlapped layers due to their high proliferation rate, showing their unfitness in biomaterial surface test. Nevertheless, hDPSC cultured inside 3D-matrices reproduced an osteocyte-like three-dimensional network, potentially useful in the repair of critical size bone defects. The behavior of the two cell models suggests a different use in biomaterial cell cultures: committed osteoblast cells could be appropriate in selecting the best surfaces to improve osseointegration, while multipotent cells could be suitable to obtain in vitro osteocyte-like network for regenerative medicine. The originality of the present work consists in studying for the first time two different cell models (committed versus multipotent) compared in parallel different biomaterial cultures, thus suggesting distinct targets for each cellular model.

Optimization of culture of mesenchymal stem cells: a comparison of conventional plate and microcarrier cultures.

OBJECTIVES: There has been increasing interest in mesenchymal stem cells (MSCs) because of their potential use for regenerative therapy; however, there is no well-defined protocol for MSCs culture. This study compares techniques of conventional plate and microcarrier culturing of MSCs. METHODS AND RESULTS: Here, different conditions for isolation and expansion of rat MSCs have been examined and it was found that plating density and plating time in primary culture played important roles for culture of these rat MSCs. When plated at 10(8) /cm(2) density for 72 h, in primary culture, recycling stem cells (RS cells) predominated, and characteristics of rat MSCs (including morphology, growth rate, phenotype and differentiation potentials) remained stable during expansion until passage 14. For subculture of the cells, it was found that their growth rate when incubated at 33 °C was higher than those incubated at 37 °C, and maximal increase was 10- and 6-fold respectively. When cultured using microcarriers, at a density of 1 × 10(5) /mg beads, growth kinetics, phenotype and differentiation potentials also remained constant for cells between passage 2nd and 14th; their maximal number increased 16-fold. CONCLUSIONS: Compared to conventional plate culture, culture using gelatine porous microcarrier Cultispher-S was superior for large-scale production of rat MSCs.

Reconstruction of pulmonary artery with porcine small intestinal submucosa in a lamb surgical model: Viability and growth potential.

OBJECTIVES: This study investigated the time-dependent remodeling and growth potential of porcine small intestine submucosa as a biomaterial for the reconstruction of pulmonary arteries in a lamb model. METHODS: Left pulmonary arteries were partially replaced with small intestine submucosal biomaterial in 6 lambs. Two animals each were humanely killed at 1, 3, and 6 months. Computed tomographic angiography, macroscopic examination of the implanted patch, and microscopic analysis of tissue explants were performed. RESULTS: All animals survived without complications. Patency and arborization of the pulmonary arteries were detected 6 months after implantation. There was no macroscopic narrowing or aneurysm formation in the patch area. The luminal appearance of the patch was similar to the intimal layer of the adjacent native pulmonary artery. Scanning electron microscopy showed that the luminal surface of the patch was covered by confluent cells. Immunohistochemical examination confirmed endothelialization of the luminal side of the patch in all of the explanted patches. The presence of smooth muscle cells in the medial layer was confirmed at all time points; however, expression of elastin, growth of the muscular layer, and complete degradation of patch material were detectable only after 6 months. The presence of c-Kit-positive cells suggests migration of multipotent cells into the patch, which may play a role in remodeling the small intestine submucosal biomaterial. CONCLUSIONS: Our data confirmed that remodeling and growth potential of the small intestine submucosal biomaterial are time dependent. Additional experiments are required to investigate the stability of the patch material over a longer period.

Isolation of novel multipotent neural crest-derived stem cells from adult human inferior turbinate.

Adult human neural crest-derived stem cells (NCSCs) are of extraordinary high plasticity and promising candidates for the use in regenerative medicine. Here we describe for the first time a novel neural crest-derived stem cell population within the respiratory epithelium of human adult inferior turbinate. In contrast to superior and middle turbinates, high amounts of source material could be isolated from human inferior turbinates. Using minimally-invasive surgery methods isolation is efficient even in older patients. Within their endogenous niche, inferior turbinate stem cells (ITSCs) expressed high levels of nestin, p75(NTR), and S100. Immunoelectron microscopy using anti-p75 antibodies displayed that ITSCs are of glial origin and closely related to nonmyelinating Schwann cells. Cultivated ITSCs were positive for nestin and S100 and the neural crest markers Slug and SOX10. Whole genome microarray analysis showed pronounced differences to human ES cells in respect to pluripotency markers OCT4, SOX2, LIN28, and NANOG, whereas expression of WDR5, KLF4, and c-MYC was nearly similar. ITSCs were able to differentiate into cells with neuro-ectodermal and mesodermal phenotype. Additionally ITSCs are able to survive and perform neural crest typical chain migration in vivo when transplanted into chicken embryos. However ITSCs do not form teratomas in severe combined immunodeficient mice. Finally, we developed a separation strategy based on magnetic cell sorting of p75(NTR) positive ITSCs that formed larger neurospheres and proliferated faster than p75(NTR) negative ITSCs. Taken together our study describes a novel, readily accessible source of multipotent human NCSCs for potential cell-replacement therapy.

Vascular wall-resident CD44+ multipotent stem cells give rise to pericytes and smooth muscle cells and contribute to new vessel maturation.

Here, we identify CD44(+)CD90(+)CD73(+)CD34(-)CD45(-) cells within the adult human arterial adventitia with properties of multipotency which were named vascular wall-resident multipotent stem cells (VW-MPSCs). VW-MPSCs exhibit typical mesenchymal stem cell characteristics including cell surface markers in immunostaining and flow cytometric analyses, and differentiation into adipocytes, chondrocytes and osteocytes under culture conditions. Particularly, TGFß1 stimulation up-regulates smooth muscle cell markers in VW-MPSCs. Using fluorescent cell labelling and co-localisation studies we show that VW-MPSCs differentiate to pericytes/smooth muscle cells which cover the wall of newly formed endothelial capillary-like structures in vitro. Co-implantation of EGFP-labelled VW-MPSCs and human umbilical vein endothelial cells into SCID mice subcutaneously via Matrigel results in new vessels formation which were covered by pericyte- or smooth muscle-like cells generated from implanted VW-MPSCs. Our results suggest that VW-MPSCs are of relevance for vascular morphogenesis, repair and self-renewal of vascular wall cells and for local capacity of neovascularization in disease processes.

Deleterious effects of freezing on osteogenic differentiation of human adipose-derived stromal cells in vitro and in vivo.

Human adipose-derived stromal cells (hASCs) represent a multipotent stromal cell type with a proven capacity to undergo osteogenic differentiation. Many hurdles exist, however, between current knowledge of hASC osteogenesis and their potential future use in skeletal tissue regeneration. The impact of frozen storage on hASC osteogenic differentiation, for example, has not been studied in detail. To examine the effects of frozen storage, hASCs were harvested from lipoaspirate and either maintained in standard culture conditions or frozen for 2 weeks under standard conditions (90% fetal bovine serum, 10% dimethyl sulfoxide). Next, in vitro parameters of cell morphology (surface electron microscopy [EM]), cell viability and growth (trypan blue; bromodeoxyuridine incorporation), osteogenic differentiation (alkaline phosphatase, alizarin red, and quantitative real-time (RT)-polymerase chain reaction), and adipogenic differentiation (Oil red O staining and quantitative RT-polymerase chain reaction) were performed. Finally, in vivo bone formation was assessed using a critical-sized cranial defect in athymic mice, utilizing a hydroxyapatite (HA)-poly(lactic-co-glycolic acid) scaffold for ASC delivery. Healing was assessed by serial microcomputed tomography scans and histology. Freshly derived ASCs differed significantly from freeze-thaw ASCs in all markers examined. Surface EM showed distinct differences in cellular morphology. Proliferation, and osteogenic and adipogenic differentiation were all significantly hampered by the freeze-thaw process in vitro (*P < 0.01). In vivo, near complete healing was observed among calvarial defects engrafted with fresh hASCs. This was in comparison to groups engrafted with freeze-thaw hASCs that showed little healing (*P < 0.01). Finally, recombinant insulin-like growth factor 1 or recombinant bone morphogenetic protein 4 was observed to increase or rescue in vitro osteogenic differentiation among frozen hASCs (*P < 0.01). The freezing of ASCs for storage significantly impacts their biology, both in vitro and in vivo. The ability of ASCs to successfully undergo osteogenic differentiation after freeze-thaw is substantively muted, both in vitro and in vivo. The use of recombinant proteins, however, may be used to mitigate the deleterious effects of the freeze-thaw process.

Assessment of DNA damage in human bone marrow cells and multipotent mesenchymal stromal cells.

We carried out a comparative analysis of DNA damage (percentage of DNA in comet tail) and frequencies of comets in apoptotic cells in BM samples and cultures of BM multipotent mesenchymal stromal cells at different terms of culturing (passages 3-11). The levels of DNA damage in mesenchymal stromal cells remained unchanged during culturing (3.5 ± 0.9 and 4.4 ± 1.2%) and did not differ from those in BM cells (3.6 ± 0.8%). In BM samples, 10-28% atypical cells with high level of DNA damage were detected. In mesenchymal stromal cells, 2.8 ± 0.9 and 3.6 ± 1.8% apoptotic cells were detected at early and late passages, respectively.

The use of cellular magnetic resonance imaging to track the fate of iron-labeled multipotent stromal cells after direct transplantation in a mouse model of spinal cord injury.

PURPOSE: The objective of this study was to track the fate of iron-labeled, multipotent stromal cells (MSC) after their direct transplantation into mice with spinal cord injuries using magnetic resonance imaging (MRI). PROCEDURES: Mice with spinal cord injuries received a direct transplant of (1) live MSC labeled with micron-sized iron oxide particles (MPIO); (2) dead, MPIO-labeled MSC; (3) unlabeled MSC; or (4) free MPIO and were imaged at 3 T for 6 weeks after transplantation. RESULTS: Live, iron-labeled MSC appeared as a well-defined region of signal loss in the mouse spinal cord at the site of transplant. However, the MR appearance of dead, iron-labeled MSC and free iron particles was similar and persisted for the 6 weeks of the study. CONCLUSIONS: Iron-labeled stem cells can be detected and monitored in vivo after direct transplantation into the injured spinal cord of mice. However, the fate of the iron label is not clear. Our investigation indicates that caution should be taken when interpreting MR images after direct transplantation of iron-labeled cells.

Differential expression of biofunctional GM1 and GM3 gangliosides within the plastic-adherent multipotent mesenchymal stromal cell population.

BACKGROUND AIMS: It is unclear whether the plastic-adherent multipotent mesenchymal stromal cells (MSC) isolated from human bone marrow (BM) represent a uniform cell population or are heterogeneous in terms of cell-surface constituents and hence functionality. METHODS: We investigated the expression profile of certain biofunctional lipids by plastic-adherent MSC, focusing particularly on two membrane microdomain (lipid raft)-associated monosialogangliosides, GM1 and GM3, using indirect confocal laser scanning fluorescence microscopy and flow cytometry. RESULTS: Phenotypically, we observed a differential expression where certain MSC subsets exhibited GM1, GM3 or both at the plasma membrane. Furthermore, disialoganglioside GD2 detection increased the complexity of the expression patterns, giving rise to seven identifiable cell phenotypes. Variation of standard culture conditions, such as the number of cell passage and period in culture, as well as donors, did not influence the heterologous ganglioside expression profile. In contrast, the binding of various lectins appeared homogeneous throughout the MSC population, indicating that the general glycosylation pattern remained common. Morphologically, the expression of a given ganglioside-based phenotype was not related to a cell with particular size or shape. Interestingly, a segregation of GM1 and GM3 clusters was observed, GM3 being mostly excluded from the highly curved plasma membrane protrusions. CONCLUSIONS: These data highlight the phenotypic heterogeneity of plastic-adherent MSC in terms of certain lipid constituents of the plasma membrane, and the presence and/or absence of distinct ganglioside-based membrane microdomains suggest their potential functional diversity.

Engineered extracellular matrices modulate the expression profile and feeder properties of bone marrow-derived human multipotent mesenchymal stromal cells.

The bone marrow harbors multipotent mesenchymal stromal cells (MSCs) that nurture hematopoietic stem cells (HSCs). The extracellular matrix (ECM) is an integral part of the bone marrow, and the aim of this study was therefore to examine the effect of engineered ECM substrates on MSC gene expression over time and to determine quantitatively the functional ability of ECM-cultured MSCs to support HSCs. ECMs were surface immobilized using thin films of maleic anhydride to covalently immobilize tropocollagen or fibrillar collagen type I to the substrate. Where indicated, collagen type I fibrils were supplemented with heparin or hyaluronic acid. All surfaces maintained MSC viability and supported cell expansion. Microarray analysis of MSCs cultured on engineered ECM substrates revealed that culture time, as well as substrate composition, significantly affected expression levels. Based on these studies, it was possible to predict the effect of these substrates on in vitro HSC clonogenicity and self-renewal. The ability to regulate the expression of stromal factors using engineered ECM is exciting and warrants further studies to identify the ECM components and combinations that maximize the expansion of clonogenic HSCs.