hMSCs In Culture Research Articles

Fully Automated Cultivation of Adipose-Derived Stem Cells in the StemCellDiscovery—A Robotic Laboratory for Small-Scale, High-Throughput Cell Production Including Deep Learning-Based Confluence Estimation

Laboratory automation is a key driver in biotechnology and an enabler for powerful new technologies and applications. In particular, in the field of personalized therapies, automation in research and production is a prerequisite for achieving cost efficiency and broad availability of tailored treatments. For this reason, we present the StemCellDiscovery, a fully automated robotic laboratory for the cultivation of human mesenchymal stem cells (hMSCs) in small scale and in parallel. While the system can handle different kinds of adherent cells, here, we focus on the cultivation of adipose-derived hMSCs. The StemCellDiscovery provides an in-line visual quality control for automated confluence estimation, which is realized by combining high-speed microscopy with deep learning-based image processing. We demonstrate the feasibility of the algorithm to detect hMSCs in culture at different densities and calculate confluences based on the resulting image. Furthermore, we show that the StemCellDiscovery is capable of expanding adipose-derived hMSCs in a fully automated manner using the confluence estimation algorithm. In order to estimate the system capacity under high-throughput conditions, we modeled the production environment in a simulation software. The simulations of the production process indicate that the robotic laboratory is capable of handling more than 95 cell culture plates per day.

Agitation in a microcarrier-based spinner flask bioreactor modulates homeostasis of human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) are well known in cell therapy due to their secretion of trophic factors, multipotent differentiation potential, and ability for self-renewal. As a result, the number of clinical trials has been steadily increasing over the last decade highlighting the need for in vitro systems capable of producing large quantities of cells to meet growing demands. However, hMSCs are highly sensitive to microenvironment conditions, including shear stress caused by dynamic bioreactor systems, and can lead to alteration of cellular homeostasis. In this study, hMSCs were expanded on microcarriers within a 125 mL spinner flask bioreactor system. Our results demonstrate a three-fold expansion over seven days. Furthermore, our results show that culturing hMSCs in the microcarrier-based suspension bioreactor (compared to static planar culture) results in smaller cell size and higher levels of reactive oxidative species (ROS) and ROS regulator Sirtuin-3, which have implications on the nicotinamide adenine dinucleotide metabolic pathway and metabolic homeostasis. In addition, hMSCs in the bioreactor showed the increased Prostaglandin E2 secretion as well as reduced the Indoleamine-pyrrole 2,3-dioxygenase secretion upon stimulus with interferon gamma. The results of this study provide understanding of potential hMSC physiology alterations impacted by bioreactor microenvironment during scalable production of hMSCs for biomanufacturing and clinical trials.

Interaction of Human Mesenchymal Stem Cells with Soft Nanocomposite Hydrogels Based on Polyethylene Glycol and Dendritic Polyglycerol

AbstractKeeping the stemness of human mesenchymal stem cells (hMSCs) and their adipocyte differentiation potential is critical for clinical use. However, these features are lost on traditional substrates. hMSCs have often been studied on stiff materials whereas culturing hMSCs in their native niche increases their potential. Herein, a patterned hydrogel nanocomposite with the stiffness of liver tissues is obtained without any molding process. To investigate hMSCs' mechanoresponse to the material, the RGD spacing units and the stiffness of the hydrogels are dually tuned via the linker length. This work suggests that hMSCs' locomotion is influenced by the nature of the hydrogel layer (bulk or thin film). Contrary to on bulk surfaces, cell traction occurs during cell spreading on thin films. In addition, hMSCs' spreading behavior varies from shorter to longer linker‐based hydrogels, where on both surfaces hMSCs maintains their stemness as well as their adipogenic differentiation potential with a higher number of adipocytes for nanocomposites with a longer polymer linker. Overall, this work addresses the need for a new alternative for hMSCs culture allowing the cells to differentiate exclusively into adipocytes. This material represents a cell‐responsive platform with a tissue‐mimicking architecture given by the mechanical and morphological properties of the hydrogel.

Sulfonated Polyrotaxane Surfaces with Basic Fibroblast Growth Factor Alter the Osteogenic Potential of Human Mesenchymal Stem Cells in Short-Term Culture.

Human mesenchymal stem cells (hMSCs) are prone to senescence and lose their differentiation potential when expanded under nonfavorable conditions. This leads to the underutilization of hMSCs in clinical situations such as bone regeneration. The use of growth factors and small molecules as supplements and changing the physical properties of the cell culture surface have been explored to maintain the self-renewal and differentiation potential of hMSCs during the in vitro expansion phase. Here, we have explored the effect of polyrotaxanes (PRXs) with different molecular mobilities along with either soluble or immobilized fibroblast growth factor 2 (FGF2) in the maintenance of the osteogenic differentiation potential of hMSCs during in vitro expansion. We found that a less expanded shape of the hMSCs was associated with highly mobile PRX surfaces, and less mobile PRX surfaces led to flattened cell morphology. The presence of FGF2 induced further expansion of the cell shape and size. The immobilization of FGF2 helped to improve the yield of hMSCs on highly mobile surfaces by promoting cell attachment to the surfaces. hMSCs cultured on highly mobile PRX surfaces exhibited poor actin cytoskeletal organization and retention of the transcriptional regulator, yes-associated protein (YAP), in cytoplasm in contrast to the hMSCs on less mobile PRX surfaces. When the hMSCs that proliferated under these conditions were collected and subjected to osteogenic differentiation on tissue culture polystyrene (TCPS) surfaces, we found that only the hMSCs cultured on highly mobile PRXs with FGF2 in both soluble and immobilized forms showed mineralization indicative of osteogenic differentiation. Further, we found that hMSCs cultured on highly mobile PRX surfaces expressed higher levels of stemness marker genes, Nanog and Oct4. These results indicate that culturing hMSCs on PRX surfaces with different molecular mobilities even for a short period of time (4 days) was sufficient to cause a drastic change in the osteogenic potential. From these results, it is suggested that apart from the use of supplements such as FGF2 in its freely soluble or immobilized form, the consideration of proper molecular mobility of the substrates could enable us to design better culture conditions for the hMSCs with osteogenic potential.

Administrations of human adult ischemia-tolerant mesenchymal stem cells and factors reduce amyloid beta pathology in a mouse model of Alzheimer's disease

The impact of human adult ischemia-tolerant mesenchymal stem cells (hMSCs) and factors (stem cell factors) on cerebral amyloid beta (Aβ) pathology was investigated in a mouse model of Alzheimer's disease (AD). To this end, hMSCs were administered intravenously to APPPS1 transgenic mice that normally develop cerebral Aβ. Quantitative reverse transcriptase polymerase chain reaction biodistribution revealed that intravenously delivered hMSCs were readily detected in APPPS1 brains 1 hour following administration, and dropped to negligible levels after 1 week. Notably, intravenously injected hMSCs that migrated to the brain region were localized in the cerebrovasculature, but they also could be observed in the brain parenchyma particularly in the hippocampus, as revealed by immunohistochemistry. A single hMSC injection markedly reduced soluble cerebral Aβ levels in APPPS1 mice after 1 week, although increasing several Aβ-degrading enzymes and modulating a panel of cerebral cytokines, suggesting an amyloid-degrading and anti-inflammatory impact of hMSCs. Furthermore, 10 weeks of hMSC treatment significantly reduced cerebral Aβ plaques and neuroinflammation in APPPS1 mice, without increasing cerebral amyloid angiopathy or microhemorrhages. Notably, a repeated intranasal delivery of soluble factors secreted by hMSCs in culture, in the absence of intravenous hMSC injection, was also sufficient to diminish cerebral amyloidosis in the mice. In conclusion, this preclinical study strongly underlines that cerebral amyloidosis is amenable to therapeutic intervention based on peripheral applications of hMSC or hMSC factors, paving the way for a novel therapy for Aβ amyloidosis and associated pathologies observed in AD.

Neurogenesis Is Induced by Electrical Stimulation of Human Mesenchymal Stem Cells Co-Cultured With Mature Neuronal Cells.

For electrical stimulation of hMSCs, gold nanoparticles were coated onto polyethyleneimine coated glass cover slips. The effects of pulsed or constant electrical stimulation upon cytotoxicity and differentiation of hMSCs were examined. The effects of co culturing hMSCs with neuronal cells were also tested. The neuronal differentiation of the stem cells was evaluated by determining the expression of neuron-specific genes and proteins using RT-PCR and Western blotting. Morphological changes were evaluated by scanning electron microscopy. The hMSCs co-cultured with mature neuronal cells and stimulated with electrical shock showed the greatest level of neurite outgrowth (>150 mm) and smaller cell body sizes.

Oscillating Magnet Array-Based Nanomagnetic Gene Transfection of Human Mesenchymal Stem Cells

In this work, the potential of nanomagnetic transfection of primary human mesenchymal stem cells (hMSCs) and the effects of a novel nonviral oscillating magnet array system in enhancing transfection efficiency were investigated. Green fluorescent protein plasmids coupled to magnetic nanoparticles (MNPs) were introduced onto hMSCs in culture. Magnetic fields generated by arrays of neodymium iron boron magnets positioned below the culture plates direct the MNP/DNA complexes into contact with the cells. The magnet arrays were oscillated, promoting more efficient endocytosis via mechanical stimulation. Green fluorescent protein expression, cell viability and stem cell surface markers were assayed. MNP/DNA complexes were delivered into hMSCs, and the oscillating magnet array system appears to improve transfection efficiency as well as cell viability. The expression of hMSC-specific cell surface markers was unaffected. Nonviral transfection using MNPs and oscillating magnet arrays offers a more efficient and 'cell-friendly' method of transfecting hMSCs than other nonviral techniques, while preserving their stem cell characteristics.

Directed differentiation of human mesenchymal stem cells toward a cardiomyogenic fate commitment through formation of cell aggregates

Cell morphology is known to modulate the multipotential lineage commitment of stem cells. We provide a new strategy to induce the early lineage commitment of human mesenchymal stem cells (hMSCs) toward a cardiomyogenic fate through the formation of cell aggregates. A surface-immobilized polyamidoamine dendrimer with fifth generation of dendron structure was used during the culturing of hMSCs. These hMSCs cultured on the G5 surface formed aggregates through active migration and division. More than 22% of cardiac troponin-T (cTnT)-positive (cTnT+) cells in aggregates formed on the dendrimer surface; the population formed on the dendrimer surface was higher than that in conventional culture vessel. When cell aggregate was reseeded onto a fresh G5 surface, single cells migrated out of the aggregates, proliferated, and formed new aggregates. This passage method, accompanied with repetitive aggregate dispersion and formation, was applied to cultures over 40 days. The proportion of cTnT+ cells increased to 62% by the end of third passage. Our results suggest that culturing hMSCs on G5 surface results in directed commitment of the hMSCs toward a cardiomyocyte-like fate.

Preactivation of Human MSCs with TNF-α Enhances Tumor-Suppressive Activity

Mesenchymal stem/stromal cells (MSCs) can either suppress or promote tumors. We found previously that incubation of human bone marrow MSCs (hMSCs) with TNF-α upregulated multiple genes including TRAIL, which has cancer apoptotic activity. Here, we show that weekly infusions into mice of hMSCs preactivated with TNF-α inhibited the progression of lung tumors formed from MDA-MB-231 breast cancer cells (MDA). In coculture, preactivated hMSCs induced apoptosis in MDA and several other TRAIL-sensitive cancer cell lines. TRAIL was further upregulated by apoptotic MDA cells in a TLR3-dependent manner; this feedforward cycle increased MDA cell apoptosis, and the chemotherapeutic drug doxorubicin had a synergistic effect. Also, activated hMSCs secreted DKK3 to suppress MDA cell cycling, leading to a decrease in β-catenin and cyclin D1/D3 and an increase in p21. Thus, culturing hMSCs with TNF-α enhances their tumor-suppressive properties and may represent a useful strategy to develop hMSC-based approaches for the treatment of cancer.

Generation of neural stem cell-like cells from bone marrow-derived human mesenchymal stem cells

Under appropriate culture conditions, bone marrow (BM)-derived mesenchymal stem cells are capable of differentiating into diverse cell types unrelated to their phenotypical embryonic origin, including neural cells. Here, we report the successful generation of neural stem cell (NSC)-like cells from BM-derived human mesenchymal stem cells (hMSCs). Initially, hMSCs were cultivated in a conditioned medium of human neural stem cells. In this culture system, hMSCs were induced to become NSC-like cells, which proliferate in neurosphere-like structures and express early NSC markers. Like central nervous system-derived NSCs, these BM-derived NSC-like cells were able to differentiate into cells expressing neural markers for neurons, astrocytes, and oligodendrocytes. Whole-cell patch clamp recording revealed that neuron-like cells, differentiated from NSC-like cells, exhibited electrophysiological properties of neurons, including action potentials. Transplantation of NSC-like cells into mouse brain confirmed that these NSC-like cells retained their capability to differentiate into neuronal and glial cells in vivo. Our data show that multipotent NSC-like cells can be efficiently produced from BM-derived hMSCs in culture and that these cells may serve as a useful alternative to human neural stem cells for potential clinical applications such as autologous neuroreplacement therapies.

Osteoinduction of Human Mesenchymal Stem Cells by Bioactive Composite Scaffolds without Supplemental Osteogenic Growth Factors

The development of a new family of implantable bioinspired materials is a focal point of bone tissue engineering. Implant surfaces that better mimic the natural bone extracellular matrix, a naturally nano-composite tissue, can stimulate stem cell differentiation towards osteogenic lineages in the absence of specific chemical treatments. Herein we describe a bioactive composite nanofibrous scaffold, composed of poly-caprolactone (PCL) and nano-sized hydroxyapatite (HA) or beta-tricalcium phosphate (TCP), which was able to support the growth of human bone marrow mesenchymal stem cells (hMSCs) and guide their osteogenic differentiation at the same time. Morphological and physical/chemical investigations were carried out by scanning, transmission electron microscopy, Fourier-transform infrared (FTIR) spectroscopy, mechanical and wettability analysis. Upon culturing hMSCs on composite nanofibers, we found that the incorporation of either HA or TCP into the PCL nanofibers did not affect cell viability, meanwhile the presence of the mineral phase increases the activity of alkaline phosphatase (ALP), an early marker of bone formation, and mRNA expression levels of osteoblast-related genes, such as the Runt-related transcription factor 2 (Runx-2) and bone sialoprotein (BSP), in total absence of osteogenic supplements. These results suggest that both the nanofibrous structure and the chemical composition of the scaffolds play a role in regulating the osteogenic differentiation of hMSCs.

Immunomodulative Efficacy of Bone Marrow-Derived Mesenchymal Stem Cells Cultured in Human Platelet Lysate

Human mesenchymal stem cells (hMSCs) are considered to be a promising tool for novel cell-based therapies. Clinical applications in solid organ transplantation were hampered by the dependence on animal serum for hMSCs clinical scale expansion until substitution with human platelet lysate (HPL) became a promising alternative. Therefore we focused on a direct comparison of immunomodulatory properties of hMSCs cultured in HPL or fetal calf serum (FCS). Phenotypic characterization, detection of cytokine secretion and effects on alloantigen- and mitogen-induced lymphocyte proliferation as well as degranulation of cytomegalovirus-specific cytotoxic T cells were applied in potency assays. We demonstrated that HPL-cultured MSCs have comparable immunomodulatory capacities to their FCS-cultured counterparts. The observed immunomodulatory properties include a beneficial inhibitory effect on immune cell proliferation and an unaffected viral T cell immunity. Thus, culturing hMSCs in HPL generates an efficient and safe expansion combined with intriguing immunomodulatory properties making these cells an attractive cell therapeutic tool.

The Cdc42 Guanine Nucleotide Exchange Factor FGD1 Regulates Osteogenesis in Human Mesenchymal Stem Cells

Loss of function mutations in FGD1 result in faciogenital dysplasia, an X-linked human developmental disorder that adversely affects the formation of multiple skeletal structures. FGD1 encodes a guanine nucleotide exchange factor that specifically activates Cdc42, a Rho family small GTPase that regulates a variety of cellular behaviors. We have found that FGD1 is expressed in human mesenchymal stem cells (hMSCs) isolated from adult bone marrow. hMSCs are multipotent cells that can differentiate into many cell types, including fibroblasts, osteoblasts, adipocytes, and chondrocytes, and are thought to play a role in maintaining musculoskeletal tissues throughout life. We demonstrate an active role of FGD1 in osteogenic differentiation of hMSCs. During osteogenic differentiation of hMSCs in culture, we observed up-regulation of both FGD1 expression and Cdc42 activity. Activating FGD1/Cdc42 signaling by overexpression of either FGD1 or constitutively active Cdc42 promoted hMSC osteogenesis, while inhibiting Cdc42 signaling by either dominant negative mutants of FGD1 or Cdc42 suppressed osteogenesis. These results demonstrate an important role for FGD1/Cdc42 signaling in hMSC osteogenesis and suggest that the defects in bone remodeling in faciogenital dysplasia may persist throughout adult life and serve as a potential pathway that may be targeted for enhancing bone regeneration.

SOX2 Is Required for Adult Human Müller Stem Cell Survival and Maintenance of Progenicity In Vitro

SOX2, a high-mobility group transcription factor, is expressed by retinal progenitors during development. It has been associated with the ability of progenitor cells to differentiate into retinal neurons and is highly expressed by human Müller stem cells (hMSCs) in culture. The authors investigated the role of this factor in the maintenance of progenicity and neural differentiation of hMSCs in vitro. SOX2 silencing was induced by transfection of hMSCs in culture with two pGSU6-GFP SOX2 silencing constructs and a scrambled control vector. Silencing was confirmed by examination of gene and protein expression coding for SOX2. Effects of SOX2 downregulation were investigated by expression of proliferation (Ki67) and apoptotic (TUNEL, caspase) cell markers and by the expression of markers of retinal neurons (HuD, βIII tubulin, rhodopsin, BRN3B, ISL1), glia (vimentin), and the progenitor marker PAX6. SOX2 silencing caused hMSCs to rapidly adopt a neural-like morphology and was accompanied by the upregulation of specific markers of retinal neurons, including βIII tubulin, rhodopsin, BRN3B, and ISL1, and by the downregulation of the neural progenitor marker PAX6 and the glial cell marker vimentin. Interestingly, SOX2 silencing induced apoptosis, suggesting a crucial role of this factor on hMSC survival in vitro. These in vitro results parallel that seen when Sox2 is silenced in neural stem cells of lower species during development, and they suggest that Sox2 may have an important role in adult hMSC differentiation into retinal neurons in vitro.

Growth factor-defined culture medium for human mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (hMSCs) are potential cellular sources of therapeutic stem cells as they have the ability to proliferate and differentiate into a wide array of mesenchymal cell types such as osteoblasts, chondroblasts and adipocytes. hMSCs have been used clinically to treat patients with graft vs. host disease, osteogenesis imperfect, or alveolar cleft, suggesting that transplantation of hMSCs is comparatively safe as a stem cell-based therapy. However, conventional culture medium for hMSCs contains fetal bovine serum (FBS). In the present study, we developed a growth factor-defined, serum-free medium for culturing hMSCs. Under these conditions, TGF-beta1 promoted proliferation of hMSCs. The expanded hMSC population expressed the human pluripotency markers SSEA-3, -4, NANOG, OCT3/4 and SOX2. Furthermore, double positive cells for SSEA-3 and a mesenchymal cell marker, CD105, were detected in the population. The potential to differentiate into osteoblasts and adipocytes was confirmed. This work provides a useful tool to understand the basic biological properties of hMSCs in culture.

The treatment of TBI with human marrow stromal cells impregnated into collagen scaffold: Functional outcome and gene expression profile

We have previously demonstrated that human marrow stromal cells (hMSCs) embedded in collagen I scaffolds significantly enhance the restorative therapeutic effect of hMSCs after traumatic brain injury (TBI). In this study, we test the hypothesis that the collagen scaffold alters gene expression in hMSCs and that hMSCs impregnated into scaffolds increase the astrocytic expression of vascular endothelial growth factor (VEGF) in the injured brain. Following TBI induced by controlled cortical impact injury, scaffold with hMSCs (3.0×106), hMSCs-only and saline were implanted into the lesion cavity one week after brain injury (n=8/each group). Morris water maze and modified neurological severity scores were performed to evaluate the spatial learning and sensorimotor functions, respectively. Lesion volume and expression of VEGF were measured one week after different treatments. In vitro, total RNA from hMSCs was extracted one week after culture with or without collagen I scaffold for evaluation of gene microarrays. Furthermore, an RT-PCR study on a select subgroup of genes was performed to identify the changes of expression between the culturing hMSCs with collagen scaffolds and hMSCs only. The treatment of TBI with collagen scaffold impregnated with hMSCs significantly decreases the functional deficits from TBI within 7days after treatment, and significantly enhances the VEGF expression of astrocytes in the injured brain compared to the hMSCs-only group. In vitro data indicate that collagen scaffolds stimulate hMSCs to express multiple factors which may contribute to hMSC survival, tissue repair and functional recovery after TBI.

Low Oxygen Tension and Synthetic Nanogratings Improve the Uniformity and Stemness of Human Mesenchymal Stem Cell Layer

A free-standing, robust cell sheet comprising aligned human mesenchymal stem cells (hMSCs) offers many interesting opportunities for tissue reconstruction. As a first step toward this goal, a confluent, uniform hMSC layer with a high degree of alignment and stemness maintenance needs to be created. Hypothesizing that topographical cue and a physiologically relevant low-oxygen condition could promote the formation of such an hMSC layer, we studied the culture of hMSCs on synthetic nanogratings (350 nm width and 700 nm pitch) and either under 2 or 20% O(2). Culturing hMSCs on the nanogratings highly aligned the cells, but it tended to create patchy layers and accentuate the hMSC differentiation. The 2% O(2) improved the alignment and uniformity of hMSCs, and reduced their differentiation. Over a 14-day culture period, hMSCs in 2% O(2) showed uniform connexon distribution, secreted abundant extracellular matrix (ECM) proteins, and displayed a high progenicity. After 21-day culture on nanogratings, hMSCs exposed to 2% O(2) maintained a higher viability and differentiation capacity. This study established that a 2% O(2) culture condition could restrict the differentiation of hMSCs cultured on nanopatterns, thereby setting the foundation to fabricate a uniformly aligned hMSC sheet for different regenerative medicine applications.

Ultrasound induced mechanical induction of mesenchymal stem cells.

Low-intensity pulsed ultrasound (LIPUS) has been used to accelerate fracture healing and tissue regeneration but the biological mechanism of these responses is not completely understood. Stem cell activity can be induced through biochemical and mechanical mechanisms. Despite the common use of LIPUS in fracture healing and tissue regeneration, there are only a few studies that examine the mechanical induction of stem cells with ultrasound. The purpose of this study is to determine the effects of ultrasound-generated mechanical stimulus on the behavior of human mesenchymal stem cells (hMSCs) in vitro. In our preliminary studies low-intensity pulsed ultrasound was used to induce mechanical strains on hMSCs in vitro. Amplitudes, pulse durations, and pulse repetition frequencies were varied such that different radiation pressures were generated on hMSCs in culture. Results indicated a significant increase in cell proliferation after 4 consecutive days of treatment as well as a significant difference in the cellular response between treatment parameters. Results suggest that LIPUS can be used to influence mechanical mediated stem cell behavior; however, more research is needed to fully elucidate the relationship between ultrasound and hMSC response.

QuantifyingIn VitroGrowth and Metabolism Kinetics of Human Mesenchymal Stem Cells Using a Mathematical Model

Better quantitative understanding of human mesenchymal stem cells (hMSCs) metabolism is needed to identify, understand, and subsequently optimize the processes in expansion of hMSCs in vitro. For this purpose, we analyzed growth of hMSCs in vitro with a mathematical model based on the mass balances for viable cell numbers, glucose, lactate, glutamine, and glutamate. The mathematical modeling had two aims: (1) to estimate kinetic parameters of important metabolites for hMSC monolayer cultures, and (2) to quantitatively assess assumptions on growth of hMSCs. Two cell seeding densities were used to investigate growth and metabolism kinetics of MSCs from three human donors. We analyzed growth up to confluency and used metabolic assumptions described in literature. Results showed a longer initial phase, a slower growth rate, and a higher glucose, lactate, glutamine, and glutamate metabolic rates at the lower cell seeding density. Higher metabolic rates could be induced by a lower contact inhibition effect when seeding at 100 cells/cm2 than when seeding at 1000 cells/cm2. In addition, parameter estimation describing kinetics of hMSCs in culture, depending on the seeding density, showed doubling times in the order of 17-32h, specific glucose consumption in the order of 1.25 x 10(-1) to 3.77 x 10(-1) pmol/cell/h, specific lactate production in the order of 2.48 x 10(-1) to 7.67 x 10(-1)pmol/cell/h, specific glutamine production in the order of 7.04 x 10(-3) to 2.27 pmol/cell/h, and specific glutamate production in the order of 4.87 x 10(-1) to 23.4 pmol/cell/h. Lactate-to-glucose yield ratios confirmed that hMSCs use glucose via anaerobic glycolysis. In addition, glutamine and glutamate metabolic shifts were identified that could be important for understanding growth of hMSCs in vitro. This study showed that the mathematical modeling approach supports quantitative analysis of important mechanisms in proliferation of hMSCs in vitro.

Multipotent human stromal cells improve cardiac function after myocardial infarction in mice without long-term engraftment

The aim of this study was to determine whether intravenously administered multipotent stromal cells from human bone marrow (hMSCs) can improve cardiac function after myocardial infarction (MI) without long-term engraftment and therefore whether transitory paracrine effects or secreted factors are responsible for the benefit conferred. hMSCs were injected systemically into immunodeficient mice with acute MI. Cardiac function and fibrosis after MI in the hMSC-treated group were significantly improved compared with controls. However, despite the cardiac improvement, there was no evident hMSC engraftment in the heart 3 weeks after MI. Microarray assays and ELISAs demonstrated that multiple protective factors were expressed and secreted from the hMSCs in culture. Factors secreted by hMSCs prevented cell death of cultured cardiomyocytes and endothelial cells under conditions that mimicked tissue ischemia. The favorable effects of hMSCs appear to reflect the impact of secreted factors rather than engraftment, differentiation, or cell fusion.