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MG53 is an important membrane repair protein and partially protects bone marrow multipotent adult progenitor cells (MAPCs) against oxidized low-density lipoprotein (ox-LDL). The present study was to test the hypothesis that the limited protective effect of MG53 on MAPCs was due to ox-LDL-induced reduction of MG53. MAPCs were cultured with and without ox-LDL (0-20 μg/mL) for up to 48 hours with or without MG53 and antioxidant N-acetylcysteine (NAC). Serum MG53 level was measured in ox-LDL-treated mice with or without NAC treatment. Ox-LDL induced significant membrane damage and substantially impaired MAPC survival with selective inhibition of Akt phosphorylation. NAC treatment effectively prevented ox-LDL-induced reduction of Akt phosphorylation without protecting MAPCs against ox-LDL. While having no effect on Akt phosphorylation, MG53 significantly decreased ox-LDL-induced membrane damage and partially improved the survival, proliferation and apoptosis of MAPCs in vitro. Ox-LDL significantly decreased MG53 level in vitro and serum MG53 level in vivo without changing MG53 clearance. NAC treatment prevented ox-LDL-induced MG53 reduction both in vitro and in vivo. Combined NAC and MG53 treatment significantly improved MAPC survival against ox-LDL. These data suggested that NAC enhanced the protective effect of MG53 on MAPCs against ox-LDL through preventing ox-LDL-induced reduction of MG53., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Background Aims: Myelodysplastic syndromes (MDS) are a group of clonal stem cell disorders affecting the normal hematopoietic differentiation process and leading to abnormal maturation and differentiation of all blood cell lineages. Treatment options are limited, and there is an unmet medical need for effective therapies for patients with severe cytopenias., Methods: We demonstrate that multipotent adult progenitor cells (MAPC) improve the function of hematopoietic progenitors derived from human MDS bone marrow (BM) by significantly increasing the frequency of primitive progenitors as well as the number of myeloid colonies., Results: This effect was more pronounced in a non-contact culture, indicating the importance of soluble factors produced by the MAPC cells. Moreover, the cells did not stimulate the growth of the abnormal MDS clone, as shown by fluorescent in situ hybridization analysis on BM cells from patients with a known genetic abnormality. We also demonstrate that MAPC cells can provide stromal support for patient-derived hematopoietic cells. When MAPC cells were intravenously injected into a mouse model of MDS, they migrated to the site of injury and increased the hematopoietic function in diseased mice., Discussion: The preclinical studies undertaken here indicate an initial proof of concept for the use of MAPC cell therapy in patients with MDS-related severe and symptomatic cytopenias and should pave the way for further investigation in clinical trials., (Copyright © 2017. Published by Elsevier Inc.)

: MultiStem cells are clinical-grade multipotent adult bone marrow-derived progenitor cells (MAPCs), with extensive replication potential and broader differentiation capacity compared with mesenchymal stem cells. Human MAPCs suppress T-cell proliferation induced by alloantigens and mutually interact with allogeneic natural killer cells. In this study, the interaction between MultiStem and CD8 + cytotoxic T lymphocytes (CTLs) was addressed for the first time. In an in vitro setting, the immunogenicity of MultiStem, the susceptibility of MultiStem toward CTL-mediated lysis, and its effects on CTL function were investigated. MultiStem was nonimmunogenic for alloreactive CTL induction and was-even after major histocompatibility complex class I upregulation-insensitive to alloantigen-specific CTL-mediated lysis. Furthermore, MultiStem reduced CTL proliferation and significantly decreased perforin expression during the T-cell activation phase. As a consequence, MultiStem dose-dependently impaired the induction of CTL function. These effects of MultiStem were mediated predominantly through contact-dependent mechanisms. Moreover, MultiStem cells considerably influenced the expression of T-cell activation markers CD25, CD69, and human leukocyte antigen-DR. The MultiStem-induced CD8 - CD69 + T-cell population displayed a suppressive effect on the induction of CTL function during a subsequent mixed-lymphocyte culture. Finally, the killer activity of activated antigen-specific CTLs during their cytolytic effector phase was also diminished in the presence of MultiStem. This study confirms that these clinical-grade MAPCs are an immune-modulating population that inhibits CTL activation and effector responses and are, consequently, a highly valuable cell population for adoptive immunosuppressive therapy in diseases where damage is induced by CTLs., Significance: Because multipotent adult progenitor cells (MAPCs) are among the noteworthy adult mesenchymal stem cell populations for immune therapy and have the advantage over mesenchymal stem cells (MSCs) of large-scale manufacturing and banking potential and thus prompt availability, it is important to understand how MAPCs interact with immune cells to validate their widespread therapeutic applicability. Cytotoxic immune effector cells play a crucial role in immune homeostasis and in the pathogenesis of some autoimmune diseases. This study assessed for the first time the in vitro influence of a clinical-grade human MAPC product (MultiStem) on the cytotoxic function of CD8 + T cells (CTLs) by evaluating the immunogenicity of MAPCs and the susceptibility of MAPCs toward CTL-mediated lysis and by analyzing the mechanism of MAPC-mediated modulation of CTL functionality. These results may represent a highly relevant contribution to the current knowledge and, in combination with the results of future phase II/III trials using MultiStem, could lead to an intriguing continuation of stem cell-based research for immunotherapy., (©AlphaMed Press.)

Cell therapy with bone marrow stem cells (BMSCs) remains a viable option for tissue repair and regeneration. A major challenge for cell therapy is the limited cell survival after implantation. This study was to investigate the effect of oxidized low-density lipoprotein (ox-LDL, naturally present in human blood) on BMSC injury and the effect of MG53, a tissue repair protein, for the improvement of stem cell survival. Rat bone marrow multipotent adult progenitor cells (MAPCs) were treated with ox-LDL, which caused significant cell death as reflected by the increased LDH release to the media. Exposure of MAPCs to ox-LDL led to entry of fluorescent dye FM1-43 measured under confocal microscope, suggesting damage to the plasma membrane. Ox-LDL also generated reactive oxygen species (ROS) as measured with electron paramagnetic resonance spectroscopy. While antioxidant N-acetylcysteine completely blocked ROS production from ox-LDL, it failed to prevent ox-LDL-induced cell death. When MAPCs were treated with the recombinant human MG53 protein (rhMG53) ox-LDL induced LDH release and FM1-43 dye entry were significantly reduced. In the presence of rhMG53, the MAPCs showed enhanced cell survival and proliferation. Our data suggest that membrane damage induced by ox-LDL contributed to the impaired survival of MAPCs. rhMG53 treatment protected MAPCs against membrane damage and enhanced their survival which might represent a novel means for improving efficacy for stem cell-based therapy for treatment of diseases, especially in setting of hyperlipidemia., (© 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Aims: The present study was to investigate the effect of hydrogen peroxide (H2O2) on bone marrow stem cells and their endothelial differentiation and the underlying mechanisms in vitro., Main Methods: Rat bone marrow multipotent adult progenitor cells (MAPCs) were used as the source of bone marrow stem cells, and treated with H2O2 (with the final concentration from 0 to 50 μM) with or without N-acetylcysteine (NAC, 0.1 mM). Reactive oxygen species (ROS) was measured by electron paramagnetic resonance (EPR) and fluorescent microscope. Flow cytometry and immunoblotting were used to determine apoptosis and differentiation of MAPCs., Key Findings: H2O2 generated a significant amount of intracellular and extracellular ROS in the culture system, substantially inhibited the proliferation of MAPCs and Oct-4 expression, and induced their apoptosis in a dose-dependent manner. Exposure to H2O2 also significantly attenuated the endothelial differentiation of MAPCs with reduced expression of endothelial markers CD31 and FLK-1 as well as impaired in vitro vascular structure formation. Both intracellular and extracellular ROS production from H2O2 were blocked by NAC. NAC treatment effectively prevented H2O2-induced reduction of Oct-4 expression in the cells. However, NAC treatment only partially prevented H2O2-induced apoptosis, and inhibition of cell proliferation and endothelial differentiation of MAPCs., Significance: H2O2 exposure suppressed Oct-4 expression in MAPCs through ROS-dependent mechanism, while increasing the apoptosis of MAPCs and inhibiting their proliferation and endothelial differentiation with a mechanism partially due to ROS generation in vitro., (Copyright © 2014. Published by Elsevier Inc.)

Human multipotent adult progenitor cells (hMAPCs) are isolated from bone marrow with a more extensive expansion capacity compared to human mesenchymal stem cells (hMSCs) and with the ability to differentiate into endothelium. Like hMSCs, hMAPCs inhibit T-cell proliferation induced by alloantigens. In this study, we tested the interaction between hMAPCs and natural killer (NK) cells. We assessed the susceptibility of hMAPCs to NK cell-mediated lysis and the immunomodulation of hMAPCs on NK cell function during IL-2-driven stimulation and the cytolytic effector phase. Human MAPCs express the ligands PVR and ULBP-2/5/6, which are recognized by activating NK cell receptors. However, they also express MHC class I molecules, which induce inhibitory signals in NK cells. Freshly isolated NK cells at different effector:target ratios did not kill hMAPCs as assessed by an MTT and (51)Cr-release assay, while hMAPCs impaired the cytotoxic activity of resting NK cells against the NK-sensitive K562 leukemia cell line. By contrast, IL-2-stimulated NK cells were capable of killing hMAPCs, and preactivated NK cells were not influenced during their cytotoxic effector function against K562 cells by hMAPCs. When added during the 6-day preactivation phase with IL-2, hMAPCs dose-dependently reduced NK cell proliferation in an IDO-dependent manner, but they did not influence the induction of cytotoxic capacity by IL-2. This study indicates that human MAPCs mutually interact with NK cells.

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.

Multipotent adult progenitor cells (MAPCs) are bone marrow-derived nonhematopoietic stem cells with a broad differentiation potential and extensive expansion capacity. A comparative study between human mesenchymal stem cells (hMSCs) and human MAPCs (hMAPCs) has shown that hMAPCs have clearly distinct phenotypical and functional characteristics from hMSCs. In particular, hMAPCs express lower levels of MHC class I than hMSCs and cannot only differentiate into typical mesenchymal cell types but can also differentiate in vitro and in vivo into functional endothelial cells. The use of hMSCs as cellular immunomodulatory stem cell products gained much interest since their immunomodulatory capacities in vitro became evident over the last decade. Currently, the clinical grade stem cell product of hMAPCs is already used in clinical trials to prevent graft-versus-host disease (GVHD), as well as for the treatment of acute myocardial infarct, ischemic stroke, and Crohn's disease. Therefore, we studied the immune phenotype, immunogenicity, and immunosuppressive effect of hMAPCs in vitro. We demonstrated that hMAPCs are nonimmunogenic for T-cell proliferation and cytokine production. In addition, hMAPCs exert strong immunosuppressive effects on T-cell alloreactivity and on T-cell proliferation induced by mitogens and recall antigens. This immunomodulatory effect was not MHC restricted, which makes off-the-shelf use promising. The immunosuppressive effect of hMAPCs is partially mediated via soluble factors and dependent on indoleamine 2,3-dioxygenase (IDO) activity. At last, we isolated hMAPCs, the clinical grade stem cell product of hMAPCs, named MultiStem, and hMSCs from one single donor and observed that both the immunogenicity and the immunosuppressive capacities of all three stem cell products are comparable in vitro. In conclusion, hMAPCs have potent immunomodulatory properties in vitro and can serve as a valuable cell source for the clinical use of immunomodulatory cellular stem cell product.

1. The transcription factor Oct4 is critical to the pluripotency, self-renewal and differentiation of stem cells. The aim of the present study was to investigate the effects of high glucose (HG) on the cell cycle progression of bone marrow multipotent adult progenitor cells (MAPC) and Oct4 expression, as well as the underlying mechanisms. 2. Rat MAPC were cultured in normal (5.5 mmol/L D-glucose) and HG (25.5 mmol/L D-glucose) media for up to 14 days. L-Glucose served as a high osmolarity control. Culture in HG media substantially increased the number of cells in the G(0)/G(1) phase and decreased the number in the S phase without changing the cell population in the G(2) phase. Expression of the cell cycle regulatory protein p21CIP/WAF-1 (p21), but not that of p27KIP-1 (p27), was significantly upregulated in cells cultured in HG media. Significant increases were seen in transforming growth factor (TGF)-β1 levels in cells and MAPC-conditioned medium in the presence of HG, and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was enhanced in cells cultured in the presence of HG medium without any changes in Akt phosphorylation. 3. Neutralizing TGF-β1 antibody effectively prevented HG-induced increases in ERK1/2 phosphorylation, p21 expression and suppression of cell cycle progression of MAPC. Inhibiting ERK1/2 phosphorylation with PD98059 completely blocked HG-induced p21 expression and markedly reversed HG-induced inhibition of cell cycle progression in MAPC. The HG-induced suppression of cell cycle progression was not accompanied by inhibition of cell proliferation or Oct4 expression in these cells. 4. The data indicate that HG facilitates cell cycle arrest of rat MAPC through TGF-β1-induced activation of ERK1/2 signalling and p21 expression, and that Oct4 expression in MAPC is independent of the cell cycle and/or TGF-β1 or ERK1/2 signalling in HG medium., (© 2012 The Authors Clinical and Experimental Pharmacology and Physiology © 2012 Wiley Publishing Asia Pty Ltd.)

Aims: This study was to investigate the effect of high glucose (HG) on TGF-β1 expression and the underlying mechanisms in bone marrow stem cells., Main Methods: Rat bone marrow multipotent adult progenitor cells (MAPCs) were cultured in normal (5.5mM d-glucose) and HG media (25.5mM d-glucose) for up to 14days. l-Glucose (20mM plus 5.5mM d-glucose) was used as high osmolarity control. TGF-β1 expression was evaluated using quantitative RT-PCR, ELISA, and immunofluorescence staining for its mRNA and protein level in the cells and in the conditioned media. The expression and activation of ERK1/2 and STAT3 were examined in MAPCs cultured in HG media with Western blot., Key Findings: Measurable level of TGF-β1 was detected in the cells cultured in normal media. TGF-β1 expression was substantially increased in MAPCs after 36 h of culture in HG media with over 20-fold increase in the mRNA and 5-fold increase in protein level over control. Interestingly, ERK1/2 phosphorylation was significantly increased in MAPCs cultured in HG media, while in STAT3 (Tyr705), not STAT3 (Ser727), phosphorylation was dramatically decreased. Treatment of cells with the specific MEK1 inhibitor PD98059 or U0126 suppressed ERK1/2 phosphorylation and TGF-β1 expression, and completely restored the level of STAT3 (Tyr705) phosphorylation in MAPCs cultured in HG media. Treatment of the cells with the specific STAT3 phosphorylation inhibitor AG490 significantly blocked STAT3 (Tyr705) phosphorylation and increased TGF-β1 expression without change in ERK1/2 phosphorylation in MPACs., Significance: HG increased TGF-β1 expression through inhibition of STAT3 (Tyr705) by enhanced ERK1/2 signaling in MAPCs., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We here discuss the potency and characteristics of various adult derived adherent stem cells with special focus on multipotent adult progenitor cells (MAPC) isolated first in 2002 in our lab. We describe the potency of MAPC, our current understanding in relationship with novel insights gained in epigenetic modifications that increase cellular potency, and their possible clinical applications., (Copyright © 2011. Published by Elsevier Ltd.)

Many applications of stem cell technologies require a large quantity of cells for which scalable processes of cell expansion and differentiation are essential. Multipotent adult progenitor cells (MAPCs) are adult stem cells isolated from the bone marrow with extensive self-renewal and broad differentiation capabilities. MAPCs are typically cultured surface adherent (2D) and at low cell density, making the large surface required for cell expansion a hindrance for many applications. This study demonstrates that MAPCs can be cultivated as aggregates in an undifferentiated state for at least 16 days, as levels of a number of transcripts, including Oct4, remained similar, Oct4 protein was unchanged, and differentiation to neural progenitor, endothelial cell and hepatocyte like cells was retained. Cultivation of these aggregates in stirred bioreactor lead to a 70-fold expansion in 6 days with final cell densities of close to 10⁶/mL. Importantly, the MAPC aggregates recovered from stirred bioreactors could be differentiated to hepatocyte-like cells that expressed albumin, alpha-1-antitrypsin (AAT), and tyrosine amino transferase (TAT) transcripts and also secreted albumin and urea. This method of scalable expansion combined with differentiation of MAPCs can potentially be used for generating large numbers of MAPC and MAPC-derived differentiated cells., (© 2010 Wiley Periodicals, Inc.)

This study was to investigate the effect of oxidized low-density lipoprotein (ox-LDL) on the behaviour of bone marrow stem cells and their endothelial differentiation as well as the underlying mechanisms. Adult rat bone marrow multipotent progenitor cells (MAPCs) were incubated with ox-LDL for up to 2 weeks. Ox-LDL treatment resulted in a time- and dose-dependent reduction of MAPC population in culture through a combination of decreased cell proliferation and increased apoptosis. The expression of stem cell marker Oct-4 was significantly suppressed in MAPCs by ox-LDL in a dose- and time-dependant manner. Endothelial differentiation of MAPCs was substantially inhibited by ox-LDL with markedly decreased expression of endothelial markers vWF, Flk-1 and CD31, as well as impaired in vitro vascular structure formation. Ox-LDL-induced apoptosis and inhibition of Oct-4 expression, cell proliferation and endothelial differentiation of MAPCs were associated with significant inhibition of Akt phosphorylation. Akt overexpression in MAPCs transfected with a constitutively active Akt completely reversed the effects of ox-LDL on MAPCs including enhanced apoptosis, decreased cell proliferation, suppressed Oct-4 expression and endothelial differentiation as well as in vitro vascular structure formation. In conclusion, ox-LDL promotes apoptosis and inhibits Oct-4 expression and self-renewal of MAPCs, and impairs their endothelial differentiation via suppression of Akt signalling., (© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

This study was to test the hypothesis that oxidized low-density lipoprotein (ox-LDL) modified the behavior of bone marrow stem cells, including proliferation, Oct-4 expression, and their endothelial differentiation through reactive oxygen species (ROS) formation in vitro. Rat bone marrow multipotent adult progenitor cells (MAPCs) were treated with ox-LDL with or without the antioxidant N-acetylcysteine (NAC). Ox-LDL generated a significant amount of ROS in the culture system as measured with electron paramagnetic resonance spectroscopy, and substantially inhibited the proliferation, Oct-4 expression, and endothelial differentiation of MAPCs. ROS production from ox-LDL in the culture system was completely prevented by NAC (1 mM). NAC treatment completely restored endothelial differentiation potential of MAPCs that was diminished by low-dose ox-LDL. NAC also significantly, but not completely, reversed the inhibitory effect of ox-LDL on proliferation and Oct-4 expression in MAPCs. NAC treatment only slightly restored Akt phosphorylation impaired by ox-LDL in the cells. ROS formation was important in the action of ox-LDL on MAPCs, including Oct-4 expression, proliferation, and endothelial differentiation. However, other mechanism(s) like Akt signaling and apoptosis might also play a critical role in mediating the effect of ox-LDL on these cells.

Many potential applications of stem cells require large quantities of cells, especially those involving large organs such as the liver. For such applications, a scalable reactor system is desirable to ensure a reliable supply of sufficient quantities of differentiation competent or differentiated cells. We employed a microcarrier culture system for the expansion of undifferentiated rat multipotent adult progenitor cells (rMAPC) as well as for directed differentiation of these cells to hepatocyte-like cells. During the 4-day expansion culture, cell concentration increased by 85-fold while expression level of pluripotency markers were maintained, as well as the MAPC differentiation potential. Directed differentiation into hepatocyte-like cells on the microcarriers themselves gave comparable results as observed with cells cultured in static cultures. The cells expressed several mature hepatocyte-lineage genes and asialoglycoprotein receptor-1 (ASGPR-1) surface protein, and secreted albumin and urea. Microcarrier culture thus offers the potential of large-scale expansion and differentiation of stem cells in a more controlled bioreactor environment., (Copyright © 2010 Elsevier B.V. All rights reserved.)

This study was to determine if bone marrow multipotent adult progenitor cells (MAPCs) underwent cardiac specification and Oct-4 expression during their cardiomyocyte differentiation in vitro. MAPCs were isolated from rat bone marrow, treated with 5-azacytidine (5-aza, 1μM) for 24h, and cultured in a serum-free medium for cardiac differentiation for up to 35 days. The cells started to express early cardiac-specific genes Nkx2.5 and GATA-4 with a significant increase in their mRNA level within 24h after 5-aza treatment. Western blotting analysis and immunofluorescence staining revealed that the cardiac-specific proteins connexin-43 and troponin I were expressed in the cells 7 days after 5-aza treatment. Flow cytometry analysis demonstrated that over 37% of the cells were positive for troponin I by 35 days of differentiation, although the cells did not display spontaneous contraction. On the other hand, the undifferentiated MAPCs expressed a significant level of the stem-cell-specific marker Oct-4 that was dramatically decreased in the cells shortly after the initiation of cardiomyocyte differentiation as evaluated using real-time (RT)-polymerase chain reaction, Western blotting, immunofluorescence staining, and flow cytometry. These data indicated that MAPCs were able to effectively differentiate into cardiomyocyte-like cells after 5-aza induction in association with downregulation of Oct-4 expression.

The ultimate therapy for ischemic stroke is restoration of blood supply in the ischemic region and regeneration of lost neural cells. This might be achieved by transplanting cells that differentiate into vascular or neuronal cell types, or secrete trophic factors that enhance self-renewal, recruitment, long-term survival, and functional integration of endogenous stem/progenitor cells. Experimental stroke models have been developed to determine potential beneficial effect of stem/progenitor cell-based therapies. To follow the fate of grafted cells in vivo, a number of noninvasive imaging approaches have been developed. Magnetic resonance imaging (MRI) is a high-resolution, clinically relevant method allowing in vivo monitoring of cells labeled with contrast agents. In this study, labeling efficiency of three different stem cell populations [mouse embryonic stem cells (mESC), rat multipotent adult progenitor cells (rMAPC), and mouse mesenchymal stem cells (mMSC)] with three different (ultra)small superparamagnetic iron oxide [(U)SPIO] particles (Resovist, Endorem, Sinerem) was compared. Labeling efficiency with Resovist and Endorem differed significantly between the different stem cells. Labeling with (U)SPIOs in the range that allows detection of cells by in vivo MRI did not affect differentiation of stem cells when labeled with concentrations of particles needed for MRI-based visualization. Finally, we demonstrated that labeled rMAPC could be detected in vivo and that labeling did not interfere with their migration. We conclude that successful use of (U)SPIOs for MRI-based visualization will require assessment of the optimal (U)SPIO for each individual (stem) cell population to ensure the most sensitive detection without associated toxicity.

Multipotent adult progenitor cells (MAPCs) are adult stem cells derived from the bone marrow of mouse and rat and were described for the first time in 2002 (Jiang et al., Nature 418:41-49, 2002), and subsequently (Breyer et al., Exp Hematol 34:1596-1601, 2006; Jiang et al., Exp Hematol 30:896-904, 2002; Ulloa-Montoya et al., Genome Biol 8:R163, 2007). The capacity of rodent MAPC to differentiate at the single-cell level into some of the cell types of endoderm, mesoderm, and neuroectoderm germ layer lineages makes them promising candidates for the study of developmental processes. MAPC are isolated using adherent cell cultures and are selected based on morphology after a period of about 8-18 weeks. Here, we describe a step-by-step reproducible method to isolate rat MAPC from fetal and adult bone marrow. We elaborate on several aspects of the isolation protocol including, cell density and medium components, and methods for selecting and obtaining potential MAPC clones and their characterization.

This study was designed to investigate the role of nitric oxide (NO) in bone marrow stem cells and their differentiation into endothelial cells in vitro. Adult mouse bone marrow multipotent progenitor cells (MAPCs) were used as the source of stem cells. Oct-4 expression (both mRNA and protein) was significantly increased by up to 68.0% in MAPCs when incubated with NO donors DETA-NONOate or sodium nitroprusside (SNP) in a concentration-dependant manner (n=3, P<0.05). However, the cell proliferation was dramatically decreased by over 3-folds when treated with DETA-NONOate or SNP for 48 h (n=3, P<0.05). When MAPCs were exposed to DETA-NONOate (100 microM) for the first 48 h during differentiation, the expression (both mRNA and protein) of vWF was significantly increased at day 14 in the differentiating cells. The effects of DETA-NONOate or SNP on cell proliferation, Oct-4 expression and endothelial differentiation of MAPCs were not affected by the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one or cGMP analog 8-Br-cGMP. These data indicate that NO may regulate both the pluripotency and differentiation of MAPCs via a cGMP-independent mechanism.

Despite progress in cardiovascular research, a cure for peripheral vascular disease has not been found. We compared the vascularization and tissue regeneration potential of murine and human undifferentiated multipotent adult progenitor cells (mMAPC-U and hMAPC-U), murine MAPC-derived vascular progenitors (mMAPC-VP), and unselected murine BM cells (mBMCs) in mice with moderate limb ischemia, reminiscent of intermittent claudication in human patients. mMAPC-U durably restored blood flow and muscle function and stimulated muscle regeneration, by direct and trophic contribution to vascular and skeletal muscle growth. This was in contrast to mBMCs and mMAPC-VP, which did not affect muscle regeneration and provided only limited and transient improvement. Moreover, mBMCs participated in a sustained inflammatory response in the lower limb, associated with progressive deterioration in muscle function. Importantly, mMAPC-U and hMAPC-U also remedied vascular and muscular deficiency in severe limb ischemia, representative of critical limb ischemia in humans. Thus, unlike BMCs or vascular-committed progenitors, undifferentiated multipotent adult progenitor cells offer the potential to durably repair ischemic damage in peripheral vascular disease patients.

Background: The present study examined whether transplantation of adherent bone marrow-derived stem cells, termed pMultistem, induces neovascularization and cardiomyocyte regeneration that stabilizes bioenergetic and contractile function in the infarct zone and border zone (BZ) after coronary artery occlusion., Methods and Results: Permanent left anterior descending artery occlusion in swine caused left ventricular remodeling with a decrease of ejection fraction from 55+/-5.6% to 30+/-5.4% (magnetic resonance imaging). Four weeks after left anterior descending artery occlusion, BZ myocardium demonstrated profound bioenergetic abnormalities, with a marked decrease in subendocardial phosphocreatine/ATP (31P magnetic resonance spectroscopy; 1.06+/-0.30 in infarcted hearts [n=9] versus 1.90+/-0.15 in normal hearts [n=8; P<0.01]). This abnormality was significantly improved by transplantation of allogeneic pMultistem cells (subendocardial phosphocreatine/ATP to 1.34+/-0.29; n=7; P<0.05). The BZ protein expression of creatine kinase-mt and creatine kinase-m isoforms was significantly reduced in infarcted hearts but recovered significantly in response to cell transplantation. MRI demonstrated that the infarct zone systolic thickening fraction improved significantly from systolic "bulging" in untreated animals with myocardial infarction to active thickening (19.7+/-9.8%, P<0.01), whereas the left ventricular ejection fraction improved to 42.0+/-6.5% (P<0.05 versus myocardial infarction). Only 0.35+/-0.05% donor cells could be detected 4 weeks after left anterior descending artery ligation, independent of cell transplantation with or without immunosuppression with cyclosporine A (with cyclosporine A, n=6; no cyclosporine A, n=7). The fraction of grafted cells that acquired an endothelial or cardiomyocyte phenotype was 3% and approximately 2%, respectively. Patchy spared myocytes in the infarct zone were found only in pMultistem transplanted hearts. Vascular density was significantly higher in both BZ and infarct zone of cell-treated hearts than in untreated myocardial infarction hearts (P<0.05)., Conclusions: Thus, allogeneic pMultistem improved BZ energetics, regional contractile performance, and global left ventricular ejection fraction. These improvements may have resulted from paracrine effects that include increased vascular density in the BZ and spared myocytes in the infarct zone.

Cardiovascular disease is the leading cause of death worldwide, which has encouraged the search for new therapies that enable the treatment of patients in palliative and curative ways. In the past decade, the potential benefit of transplantation of cells that are able to substitute for the injured tissue has been studied with several cell populations, such as stem cells. Some of these cell populations, such as myoblasts and bone marrow cells, are already being used in clinical trials. The laboratory of CM Verfaillie has studied primitive progenitors, termed multipotent adult progenitor cells, which can be isolated from adult bone marrow. These cells can differentiate in vitro at the single-cell level into functional cells that belong to the three germ layers and contribute to most, if not all, somatic cell types after blastocyst injection. This remarkably broad differentiation potential makes this particular cell population a candidate for transplantation in tissues in need of regeneration. Here, we focus on the regenerative capacity of multipotent adult progenitor cells in several ischemic mouse models, such as acute and chronic myocardial infarction and limb ischemia.

For decades, in vitro expansion of transplantable hematopoietic stem cells (HSCs) has been an elusive goal. Here, we demonstrate that multipotent adult progenitor cells (MAPCs), isolated from green fluorescent protein (GFP)-transgenic mice and expanded in vitro for >40-80 population doublings, are capable of multilineage hematopoietic engraftment of immunodeficient mice. Among MAPC-derived GFP+CD45.2+ cells in the bone marrow of engrafted mice, HSCs were present that could radioprotect and reconstitute multilineage hematopoiesis in secondary and tertiary recipients, as well as myeloid and lymphoid hematopoietic progenitor subsets and functional GFP+ MAPC-derived lymphocytes that were functional. Although hematopoietic contribution by MAPCs was comparable to control KTLS HSCs, approximately 10(3)-fold more MAPCs were required for efficient engraftment. Because GFP+ host-derived CD45.1+ cells were not observed, fusion is not likely to account for the generation of HSCs by MAPCs.

Background: Recently, several populations of postnatal stem cells, such as multipotent adult progenitor cells (MAPCs), have been described that have broader differentiation ability than classical adult stem cells. Here we compare the transcriptome of pluripotent embryonic stem cells (ESCs), MAPCs, and lineage-restricted mesenchymal stem cells (MSCs) to determine their relationship., Results: Applying principal component analysis, non-negative matrix factorization and k-means clustering algorithms to the gene-expression data, we identified a unique gene-expression profile for MAPCs. Apart from the ESC-specific transcription factor Oct4 and other ESC transcripts, some of them associated with maintaining ESC pluripotency, MAPCs also express transcripts characteristic of early endoderm and mesoderm. MAPCs do not, however, express Nanog or Sox2, two other key transcription factors involved in maintaining ESC properties. This unique molecular signature was seen irrespective of the microarray platform used and was very similar for both mouse and rat MAPCs. As MSC-like cells isolated under MAPC conditions are virtually identical to MSCs, and MSCs cultured in MAPC conditions do not upregulate MAPC-expressed transcripts, the MAPC signature is cell-type specific and not merely the result of differing culture conditions., Conclusion: Multivariate analysis techniques clustered stem cells on the basis of their expressed gene profile, and the genes determining this clustering reflected the stem cells' differentiation potential in vitro. This comparative transcriptome analysis should significantly aid the isolation and culture of MAPCs and MAPC-like cells, and form the basis for studies to gain insights into genes that confer on these cells their greater developmental potency.

Smooth muscle formation and function are critical in development and postnatal life. Hence, studies aimed at better understanding SMC differentiation are of great importance. Here, we report that multipotent adult progenitor cells (MAPCs) isolated from rat, murine, porcine, and human bone marrow demonstrate the potential to differentiate into cells with an SMC-like phenotype and function. TGF-beta1 alone or combined with PDGF-BB in serum-free medium induces a temporally correct expression of transcripts and proteins consistent with smooth muscle development. Furthermore, SMCs derived from MAPCs (MAPC-SMCs) demonstrated functional L-type calcium channels. MAPC-SMCs entrapped in fibrin vascular molds became circumferentially aligned and generated force in response to KCl, the L-type channel opener FPL64176, or the SMC agonists 5-HT and ET-1, and exhibited complete relaxation in response to the Rho-kinase inhibitor Y-27632. Cyclic distention (5% circumferential strain) for 3 weeks increased responses by 2- to 3-fold, consistent with what occurred in neonatal SMCs. These results provide evidence that MAPC-SMCs are phenotypically and functionally similar to neonatal SMCs and that the in vitro MAPC-SMC differentiation system may be an ideal model for the study of SMC development. Moreover, MAPC-SMCs may lend themselves to tissue engineering applications.

Previous studies have identified Sleeping Beauty transposons as efficient vectors for nonviral gene delivery in mammalian cells. However, studies demonstrating the usefulness of transposons as gene delivery vehicles into adult stem cells are lacking. Multipotent adult progenitor cells (MAPC) are nonhematopoietic stem cells with the capacity to form most, if not all, cell types of the body and as such hold great therapeutic potential. The whole-body biodistribution and persistence of MAPC are unknown, and such data would help direct clinical applications. We have nucleofected murine MAPC with two plasmid-based Sleeping Beauty transposons encoding the red fluorescent protein (DsRed2) and firefly luciferase. Transgenic euploid MAPC clones maintained their characteristic multilineage differentiation potential in vitro. DsRed2 and luciferase expression allowed for MAPC detection in vivo and in tissue sections. To confirm that transgenesis occurred by transposition into the genome of MAPC, we mapped Sleeping Beauty transposon integration sites in two MAPC clones using splinkerette PCR. This novel dual-reporter imaging approach based on the transgenesis of MAPC with Sleeping Beauty transposons sheds light on the homing patterns of MAPC and paves the way for quantification of MAPC engraftment in real time in vivo.

The quintessential stem cell is the embryonal stem (ES) cell which has unlimited self-renewal and multipotent differentiation potential. Stem cells have also been identified in most tissues. Compared with ES cells, tissue-specific stem cells have less self-renewal ability and, although they differentiate into multiple lineages, they are not multipotent. A large number of recent published studies have suggested that tissue-specific stem cells may have the ability to generate cells of tissues from unrelated organs. We have identified a population of primitive cells in normal human, rodent, and possibly other mammalian postnatal tissues that have, at the single cell level, multipotent differentiation and extensive proliferation potential, which we named multipotent adult progenitor cell (MAPC). We will discuss studies aimed at determining whether MAPCs also exist in bone marrow of other species, whether MAPCs exist in vivo or are a culture phenomenon, to further characterize MAPCs using gene expression profiling, and effects of in vivo transplantation of MAPCs.

In the immunohistochemical analysis of the brain, tissue preparation and fixation are critical steps. During our studies of transplanting human bone marrow multipotent adult progenitor cells (hMAPCs) into the rat brain, we noticed that various methods of brain tissue preparation and fixation differentially influenced antigenic preservation in the transplants and in the host brain. Here, we report a simple, effective and reproducible method of tissue preparation and fixation that results in the immunohistochemical labeling of transplanted and host cells.

Previously we reported the characterization of multipotent adult progenitor cells (MAPCs) isolated from the bone marrow of rodents. In that study, single murine MAPCs derived from ROSA-26, beta-galactosidase (beta-Gal)-positive transgenic mice were injected into E3.5 C57/B16 mouse blastocysts. The resultant chimeric blastocysts were then implanted into pseudopregnant females and were allowed to develop naturally through birth and into adulthood. Chimeric mice were sacrificed 6 to 20 weeks after birth, and were processed for histological analysis. Beta-galactosidase activity was identified in all organs and tissues examined, and tissue-specific differentiation and engraftment was confirmed by colabeling with antibodies that recognize beta-Gal and tissue-specific markers. In the present study we have examined neural engraftment derived from the clonal expansion of a single MAPC during rodent development, and characterized the neural phenotype of MAPCs in the resultant chimeric animals. Donor cell-derived beta-Gal activity was evident throughout the brain. Double and triple immunofluorescent labeling studies revealed MAPC-derived neurons (NeuN/beta-Gal) and astrocytes (GFAP/beta-Gal) in the cortex, striatum, medial septal nucleus, hippocampus, cerebellum, substantia nigra, and thalamus. More specifically, donor-derived neurons contributed to each of the cellular layers of the cortex; the pyramidal and granule cell layers, as well as the hilus, of the hippocampus; Purkinje and granule cell layers in the cerebellum; and GABAergic cells in the caudate and putamen. This study characterizes the potential for MAPCs to differentiate into specific neuronal and glial phenotypes, and to integrate normally during development, after implantation into blastocysts, and provides additional evidence that MAPCs exhibit properties similar to embryonic stem cells.

This update discusses what is known regarding embryonic and adult tissue-derived pluripotent stem cells, including the mechanisms underlying self-renewal without senescence, differentiation in multiple cell types both in vitro and in vivo, and future potential clinical uses of such stem cells. In Section I, Dr. Lansdorp reviews the structure and function of telomerase, the enzyme that restores telomeric ends of chromosomes upon cell division, highly present in embryonic stem cells but not adult stem cells. He discusses the structure and function of telomerase and signaling pathways activated by the enzyme, with special emphasis on normal and leukemic hematopoietic stem cells. In Section II, Dr. Pera reviews the present understanding of mammalian pluripotent embryonic stem cells. He discusses the concept of pluripotentiality in its embryonic context, derivation of stem cells from embryonic or fetal tissue, the basic properties of the stem cells, and methods to produce specific types of differentiated cell from stem cells. He examines the potential applications of stem cells in research and medicine and some of the barriers that must be crossed to achieve these goals. In Section III, Dr. Verfaillie reviews the present understanding of pluripotency of adult stem cells. She discusses the concept of stem cell plasticity, a term used to describe the greater potency described by several investigators of adult tissue-derived stem cells, critically reviews the published studies demonstrating stem cell plasticity, and possible mechanisms underlying such plasticity, and examines the possible role of pluripotent adult stem cells in research and medicine.