Johanna Nystedt

Human umbilical cord blood cells do not improve sensorimotor or cognitive outcome following transient middle cerebral artery occlusion in rats

The present study investigated effects of human umbilical cord blood (HUCB) cells on sensorimotor, cognitive, and histological outcome in rats subjected to transient middle cerebral artery occlusion (MCAO). Halothane anesthetized adult male Wistar rats were subjected to transient MCAO for 2 h. HUCB cells (mononuclear 1-5x10(7) or Lin(-) cells 1-5x10(5)) were administered intravenously after 24 h recovery. The limb-placing test was performed on postoperative days 2, 4, 6, 9, 12, 16, and 20. In addition, beam-walking and cylinder tests were used to assess sensorimotor function at baseline, and on postoperative days 4, 12, and 20. Morris water-maze was used to assess cognitive performance on postoperative days 22-24. Subsequently, rats were perfused for measurement of infarct volumes and detection of HUCB cells by immunohistochemistry (MAB1281). MCAO rats showed a partial spontaneous recovery in sensorimotor function during the follow-up. However, the recovery profile was similar in MCAO controls and in MCAO rats that received HUCB cells. HUCB did not affect impaired water-maze performance of MCAO rats. Only few human nuclei-specific MAB1281-positive cells were detected in the ipsilateral hemisphere in MCAO rats that received HUCB cells. Infarct volumes did not differ between the experimental groups. A group of additional rats were used to further study biodistribution of intravenously given (111)In-oxine-labelled mononuclear HUCB cells in MCAO and sham-operated rats. SPECT imaging data indicated a high tracer uptake in the lung, liver, spleen, and kidney, but not in the brain immediately after administration or 24 h post-administration. The present study suggests that HUCB cells do not improve functional recovery or histological outcome in MCAO rats after systemic administration because of limited migration of cells in the ischemic brain.

Cell Surface Structures Influence Lung Clearance Rate of Systemically Infused Mesenchymal Stromal Cells

The promising clinical effects of mesenchymal stromal/stem cells (MSCs) rely especially on paracrine and nonimmunogenic mechanisms. Delivery routes are essential for the efficacy of cell therapy and systemic delivery by infusion is the obvious goal for many forms of MSC therapy. Lung adhesion of MSCs might, however, be a major obstacle yet to overcome. Current knowledge does not allow us to make sound conclusions whether MSC lung entrapment is harmful or beneficial, and thus we wanted to explore MSC lung adhesion in greater detail. We found a striking difference in the lung clearance rate of systemically infused MSCs derived from two different clinical sources, namely bone marrow (BM‐MSCs) and umbilical cord blood (UCB‐MSCs). The BM‐MSCs and UCB‐MSCs used in this study differed in cell size, but our results also indicated other mechanisms behind the lung adherence. A detailed analysis of the cell surface profiles revealed differences in the expression of relevant adhesion molecules. The UCB‐MSCs had higher expression levels of α4 integrin (CD49d, VLA‐4), α6 integrin (CD49f, VLA‐6), and the hepatocyte growth factor receptor (c‐Met) and a higher general fucosylation level. Strikingly, the level of CD49d and CD49f expression could be functionally linked with the lung clearance rate. Additionally, we saw a possible link between MSC lung adherence and higher fibronectin expression and we show that the expression of fibronectin increases with MSC culture confluence. Future studies should aim at developing methods of transiently modifying the cell surface structures in order to improve the delivery of therapeutic cells. STEM CELLS2013;31:317–326

Intra-arterial infusion of human bone marrow-derived mesenchymal stem cells results in transient localization in the brain after cerebral ischemia in rats

Cell therapies from various sources have been under intense research in stroke. Efficient homing of the cells to the injured brain without complications is necessary to realize the therapeutic potential of cell therapy. Intra-arterial (IA) infusion of cells bypasses the filtering organs and directs the cells to the target area more efficiently. Here we studied the biodistribution of human bone marrow-derived mesenchymal stromal/stem cells (BMMSCs) after a direct infusion into the external carotid artery (ECA) in rats. Cells, which were cultured without animal-derived agents and also treated with a proteolytic enzyme to transiently modify cell surface adhesion proteins, were infused 24 h after transient middle cerebral artery occlusion (MCAO). SPECT imaging was used immediately after cell infusion and 24 h thereafter to track (111)In-oxine-labeled BMMSC in sham-operated and MCAO rats. IA infusion of BMMSCs in rats resulted in immediate cell entrapment in the brain, but the majority of the signal disappeared during the next 24 h and relocated to the internal organs. In MCAO rats, radioactivity counts 24 h after infusion were higher in the ischemic hemisphere compared to the contralateral hemisphere. Our results showed that IA infusion through ECA is a safe and efficient administration route for BMMSCs resulting in a transient localization of cells in the rat brain.

Umbilical Cord Blood–derived Progenitor Cells Enhance Muscle Regeneration in Mouse Hindlimb Ischemia Model

Progenitor cell therapy is a potential new treatment option for ischemic conditions in the myocardium and skeletal muscles. However, it remains unclear whether umbilical cord blood (UCB)-derived progenitor cells can provide therapeutic effects in ischemic muscles and whether ex vivo gene transfer can be used for improving the effect. In this study, the use of a lentiviral vector led to efficient transduction of both UCB-derived hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). Our method resulted in a long-term transgene expression and did not alter the differentiation potential of either HSCs or MSCs. In addition, we studied the therapeutic potential of CD133+ and MSC progenitor cells transduced ex vivo with lentiviruses encoding the mature form of vascular endothelial growth factor D (VEGF-DΔNΔC) or the enhanced green fluorescent protein (eGFP) marker gene in a nude mouse model of skeletal muscle ischemia. Progenitor cells enhanced the regeneration of ischemic muscles without a detectable long-term engraftment of either CD133+ or MSC progenitor cells. Our results show that, rather than directly participating in angiogenesis or skeletal myogenesis, UCB-derived progenitor cells indirectly enhance the regenerative capacity of skeletal muscle after acute ischemic injury. However, VEGF-D gene transfer of progenitor cells did not improve the therapeutic effect in ischemic muscles.

Mitochondrial function and energy metabolism in umbilical cord blood- and bone marrow-derived mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) are an attractive choice for a variety of cellular therapies. hMSCs can be isolated from many different tissues and possess unique mitochondrial properties that can be used to determine their differentiation potential. Mitochondrial properties may possibly be used as a quality measure of hMSC-based products. Accordingly, the present work focuses on the mitochondrial function of hMSCs from umbilical cord blood (UCBMSC) cells and bone marrow cells from donors younger than 18 years of age (BMMSC <18) and those more than 50 years of age (BMMSC >50). Changes of ultrastructure and energy metabolism during osteogenic differentiation in all hMSC types were studied in detail. Results show that despite similar surface antigen characteristics, the UCBMSCs had smaller cell surface area and possessed more abundant rough endoplasmic reticulum than BMMSC >50. BMMSC <18 were morphologically more UCBMSC-like. UCBMSC showed dramatically higher mitochondrial-to-cytoplasm area ratio and elevated superoxide and manganese superoxide dismutase (MnSOD) levels as compared with BMMSC >50 and BMMSC <18. All hMSCs types showed changes indicative of mitochondrial activation after 2 weeks of osteogenic differentiation, and the increase in mitochondrial-to-cytoplasm area ratio appears to be one of the first steps in the differentiation process. However, BMMSC >50 showed a lower level of mitochondrial maturation and differentiation capacity. UCBMSCs and BMMSCs also showed a different pattern of exocytosed proteins and glycoproteoglycansins. These results indicate that hMSCs with similar cell surface antigen expression have different mitochondrial and functional properties, suggesting different maturation levels and other significant biological variations of the hMSCs. Therefore, it appears that mitochondrial analysis presents useful characterization criteria for hMSCs intended for clinical use.

Human bone marrow mesenchymal stem/stromal cells produce efficient localization in the brain and enhanced angiogenesis after intra-arterial delivery in rats with cerebral ischemia, but this is not translated to behavioral recovery

Intravascular cell therapy is a promising approach for the treatment of stroke. However, high accumulation of cells to lungs and other filtering organs is a major concern after intravenous (i.v.) cell transplantation. This can be circumvented by intra-arterial (i.a.) cell infusion, which improves homing of cells to the injured brain. We studied the effect of i.a. delivery of human bone marrow-derived mesenchymal cells (BMMSCs) on behavioral and histological outcome in rats after middle cerebral artery occlusion (MCAO). Sixty male Wistar rats were subjected to transient MCAO (60 min) or sham-operation. BMMSCs (1×10(6)) were infused into the external carotid artery on postoperative day 2 or 7. Histology performed after a 42-day follow-up did not detect any human cells (MAB1281) in the ischemic brain. Endothelial cell staining with RECA-1 revealed a significant increase in the number of blood vessels in the perilesional cortex in MCAO rats treated with cells on postoperative day 7. Behavioral recovery as assessed in three tests, sticky label, cylinder and Montoyas staircase, was not improved by human BMMSCs during the follow-up. In conclusion, human BMMSCs did not improve functional recovery in MCAO rats despite effective initial homing to the ischemic hemisphere and enhanced angiogenesis, when strict behavioral tests not affected by repeated testing and compensation were utilized.

The Isolation and Culture of Human Cord Blood-Derived Mesenchymal Stem Cells Under Low Oxygen Conditions

There is growing evidence that low oxygen conditions are beneficial for in vitro stem cell culturing. Mimicking the physiological oxygen tension of the placental stem cell niche in cell expansion can -ultimately result in more robust cell expansion. Growing evidence also suggests that hypoxic preconditioning of cells may improve therapeutic outcomes. Here we describe a scalable method that enables mesenchymal stromal cell expansion from virtually every cord blood unit, including those that would normally be disqualified from banking. In addition, the cells obtained by the described method fulfill exclusively the mesenchymal stromal cell characteristics defined by the International Society for Cellular Therapy.

Transient Proteolytic Modification of Mesenchymal Stromal Cells Increases Lung Clearance Rate and Targeting to Injured Tissue

Systemic infusion of therapeutic cells would be the most practical and least invasive method of administration in many cellular therapies. One of the main obstacles especially in intravenous delivery of cells is a massive cell retention in the lungs, which impairs homing to the target tissue and may decrease the therapeutic outcome. In this study we showed that an alternative cell detachment of mesenchymal stromal/stem cells (MSCs) with pronase instead of trypsin significantly accelerated the lung clearance of the cells and, importantly, increased their targeting to an area of injury. Cell detachment with pronase transiently altered the MSC surface protein profile without compromising cell viability, multipotent cell characteristics, or immunomodulative and angiogenic potential. The transient modification of the cell surface protein profile was sufficient to produce effective changes in cell rolling behavior in vitro and, importantly, in the in vivo biodistribution of the cells in mouse, rat, and porcine models. In conclusion, pronase detachment could be used as a method to improve the MSC lung clearance and targeting in vivo. This may have a major impact on the bioavailability of MSCs in future therapeutic regimes.

Human CMP-N-acetylneuraminic acid hydroxylase is a novel stem cell marker linked to stem cell-specific mechanisms.

Human stem cells contain substantial amounts of the xenoantigen N‐glycolylneuraminic acid (Neu5Gc), although the levels of Neu5Gc are low or undetectable in human body fluids and most other human tissues. The lack of Neu5Gc in human tissues has been previously explained by the loss of hydroxylase activity of the human CMP‐N‐acetylneuraminic acid hydroxylase (CMAH) protein caused by a genetic error in the human Cmah gene. We thus wanted to investigate whether the human redundant Cmah gene could still function in stem cell‐specific processes. In this study, we show that CMAH gene expression is significantly upregulated in the adult stem cell populations studied, both of hematopoietic and mesenchymal origin, and identify CMAH as a novel stem cell marker. The CMAH content co‐occurs with higher levels of Neu5Gc within stem cells as measured by mass spectrometric profiling. It seems that despite being enzymatically inactive, human CMAH may upregulate the Neu5Gc content of cells by enhancing Neu5Gc uptake from exogenous sources. Furthermore, exposure to exogenous Neu5Gc caused rapid phosphorylation of β‐catenin in both CMAH overexpressing cells and bone marrow‐derived mesenchymal stem cells, thereby inactivating Wnt/β‐catenin signaling. The data demonstrate the first molecular evidence for xenoantigen Neu5Gc‐induced alteration of crucial stem cell‐specific signaling systems for the maintenance of self renewal. These results add further emphasis to the crucial need for completely xenofree culturing conditions for human stem cells. STEM CELLS 2010;28:258–267

Safety and biodistribution study of bone marrow–derived mesenchymal stromal cells and mononuclear cells and the impact of the administration route in an intact porcine model

BACKGROUND AIMS Bone marrow mononuclear cells (BM-MNCs) and bone marrow-derived mesenchymal stem stromal cells (BM-MSCs) could have therapeutic potential for numerous conditions, including ischemia-related injury. Cells transplanted intravascularly may become entrapped in the lungs, which potentially decreases their therapeutic effect and increases the risk for embolism. METHODS Twelve pigs were divided into groups of 3 and received (99m)Tc- hydroxymethyl-propylene-amine-oxime-labeled autologous BM-MNCs or allogeneic BM-MSCs by either intravenous (IV) or intra-arterial (IA) transplantation. A whole body scan and single photon emission computed tomography/computed tomography (SPECT/CT) were performed 8 h later, and tissue biopsies were collected for gamma counting. A helical CT scan was also performed on 4 pigs to detect possible pulmonary embolism, 2 after IV BM-MSC injection and 2 after saline injection. RESULTS The transplantation route had a greater impact on the biodistribution of the BM-MSCs than the BM-MNCs. The BM-MNCs accumulated in the spleen and bones, irrespective of the administration route. The BM-MSCs had relatively higher uptake in the kidneys. The IA transplantation decreased the deposition of BM-MSCs in the lungs and increased uptake in other organs, especially in the liver. Lung atelectases were frequent due to mechanical ventilation and attracted transplanted cells. CT did not reveal any pulmonary embolism. CONCLUSIONS Both administration routes were found to be safe, but iatrogenic atelectasis might be an issue when cells accumulate in the lungs. The IA administration is effective in avoiding pulmonary entrapment of BM-MSCs. The cell type and administration method both have a major impact on the acute homing.