Heinrich Lannert

Human Alternatives to Fetal Bovine Serum for the Expansion of Mesenchymal Stromal Cells from Bone Marrow

Mesenchymal stromal cells (MSCs) are promising candidates for novel cell therapeutic applications. For clinical scale manufacturing, human factors from serum or platelets have been suggested as alternatives to fetal bovine serum (FBS). We have previously shown that pooled human serum (HS) and thrombin‐activated platelet releasate in plasma (tPRP) support the expansion of adipose tissue‐derived MSCs. Contradictory results with bone marrow (BM)‐derived MSCs have initiated a comprehensive comparison of HS, tPRP, and pooled human platelet lysate (pHPL) and FBS in terms of their impact on MSC isolation, expansion, differentiation, and immunomodulatory activity. In addition to conventional Ficoll density gradient centrifugation, depletion of lineage marker expressing cells (RosetteSep) and CD271+ sorting were used for BM‐MSC enrichment. Cells were cultured in medium containing either 10% FBS, HS, tPRP, or pHPL. Colony‐forming units and cumulative population doublings were determined, and MSCs were maximally expanded. Although both HS and tPRP comparable to FBS supported isolation and expansion, pHPL significantly accelerated BM‐MSC proliferation to yield clinically relevant numbers within the first two passages. MSC quality and functionality including cell surface marker expression, adipogenic and osteogenic differentiation, and immunosuppressive action were similar in MSCs from all culture conditions. Importantly, spontaneous cell transformation was not observed in any of the culture conditions. Telomerase activity was not detected in any of the cultures at any passage. In contrast to previous data from adipose tissue‐derived MSCs, pHPL was found to be the most suitable FBS substitute in clinical scale BM‐MSC expansion. STEM CELLS 2009;27:2331–2341

Lactosylceramide is synthesized in the lumen of the Golgi apparatus

Recently, synthesis of lactosylceramide has been described to occur on the cytosolic face of the Golgi [(1991) J. Biol. Chem. 266, 20907‐20912]. The reactions following in the biosynthesis of higher glycosphingolipids are known to take place in the lumen of the Golgi. For our understanding of the functional organization of the multiglycosyltransferase system of glycosphingolipid synthesis in the Golgi, the knowledge of the topology of individual reactions is a prerequisite. We have developed a simple and quick assay system for sphingolipid biosynthesis and have obtained evidence that lactosylceramide is synthesized in the lumen of the Golgi. Because lactosylceramide is generated by galactosylation of glucosylceramide which, in turn, is synthesized from ceramide and UDP‐Glc on the cytosolic surface of the Golgi apparatus, further efforts will be directed to the characterization of a glucosylceramide‐translocator in the Golgi membranes rather than a lactosylceramide‐translocator.

Damaged neuronal energy metabolism and behavior are improved by Ginkgo biloba extract (EGb 761).

Summary. The standardized extract EGb 761 from the dried green leaves of Ginkgo biloba is a complex mixture of ingredients with an uniquely broad spectrum of pharmacological activities on the central nervous system e.g. in memory enhancing properties and in the regulation of cerebral glucose/energy metabolism. To test these effects on both behavioral and metabolic brain parameters, the animal model of intracerebroventricular (icv) streptozotocin (STZ) treatment was used. To quantify the experimental data more precisely, animals that were good performers were separated from poor performers by means of the holeboard test before icv administration of STZ. Good performers only were considered for the study. After a 1-week training period on the holeboard improvement was seen in all animals in learning, memory and cognition, and the improvement was maintained over the investigation period of 12 weeks in the control group. In this group, the energy pool in the cerebral parietotemporal cortex was found to be large and the energy turnover high. After triplicate icv STZ injection, working memory (WM), reference memory (RM), and passive avoidance (PA) behavior fell off and continued to deteriorate throughout the investigation period. Otherwise there were no significant differences in locomotor activity, excluding the possibility that activity per se might have contributed to the behavioral abnormalities. These were accompanied by a permanent deficit in cerebral energy metabolism. The ongoing deterioration in behavior and the maintained deficit in cerebral energy metabolism occurring after a triplicate icv STZ injection were significantly slowed down by EGb761. The deficits in learning, memory and cognition were partially compensated, and the disturbances in cerebral energy metabolism returned to almost completely normal values. These findings underscore the beneficial effect of EGb761 that had been reported in dementia disorders.

Inhibition of the Neuronal Insulin Receptor Causes Alzheimer-like Disturbances in Oxidative/Energy Brain Metabolism and in Behavior in Adult Rats

Sporadic Alzheimer’s disease (SAD) is as yet of unknown etiology. Its pathogenesis, however, is multifactorial. Based on in vivo and post mortem findings in SAD patients the working hypothesis was forwarded that SAD is the brain type of diabetes mellitus II.1,2 To prove this hypothesis, an animal model was established in which the function of the neuronal insulin receptor was inhibited by the intracerebroventricularly (icv) injected diabetogenic substance streptozotocin (STZ). In short-term studies (3 weeks), after a single icv STZ, abnormalities in oxidative/energy metabolism, phospholipid composition of membranes, cholinergic and catecholaminergic functions and in learning and memory were found (for review see Ref. 3) To induce a long-term and progressive deterioration in behavior and in cerebral oxidative/energy metabolism such as is characteristic for SAD, a triplicate icv STZ challenge was performed. Learning, memory and cognition were investigated over a period of 2 and 3 months, and energy metabolism was studied at the end of these periods. We were interested to investigate whether or not estradiol and Ginkgo biloba extract (EGb761) exert beneficial effects on this neuronal damage. Estradiol was found to affect the CNS (for review see Ref. 4) and to regulate glycolysis in the brain,5 like insulin. EGb 761 demonstrated its therapeutic usefulness for the treatment of dementia and other cerebral insufficiences (for review see Ref. 6) which was clearly demonstrated in a long-term study in SAD.7

Effects of Estradiol (−17β) on learning, memory and cerebral energy metabolism in male rats after intracerebroventricular administration of streptozotocin

Summary. Treatment of adult rats with intracerebroventricularly (icv) injected streptozotocin (STZ) may provide a relevant animal model of chronic neuronal dysfunction that is characterized by a decrease in both the neuronal metabolism of glucose and the formation of energy. The present study was designed to evaluate whether or not rats treated with triplicate icv STZ injection would show long-term effects in learning and memory behavior as measured by means of a holeboard test, closed field activity, and passive avoidance behavior over a period of 6 weeks. For this purpose, animals with good performance were discriminated from those with poor performance by the holeboard test before icv administration of STZ. After a 1-week training period with the holeboard all animals improved their abilites in learning and memory, and the improvement was maintained over the investigation period of 6 weeks in the control group. After icv STZ working memory (WM), reference memory (RM), as well passive avoidance (PA) behavior decreased, deteriorating progressively during the investigation period. The latter were accompanied by a permanent deficit in cerebral energy metabolism. The ongoing deterioration in behavior and the sustained deficit in cerebral energy metabolism occurring after a triplicate icv STZ administration lead us to assume that this animal model may be appropriate for the investigation of mechanisms characteristic for sporadic Alzheimer disease. In this context, the effect of Estradiol-17β (E2) on behavior and energy metabolism was studied. We found that E2 slowed down the icv STZ-induced deterioration in memory functions, partially compensated the learning deficit, and improved the disturbances in cerebral energy metabolism to the extent that it was almost completely normal again. These findings underscore the beneficial effect of E2 in dementia disorders.

Long-term effects of corticosterone on behavior, oxidative and energy metabolism of parietotemporal cerebral cortex and hippocampus of rats: comparison to intracerebroventricular streptozotocin

We studied the effect of long-term application of corticosterone (CORT) s.c. the equivalent of cortisol in rats, on behavior, oxidative and energy metabolism in brain parietotemporal cortex and hippocampus of 1-year-old male Wistar rats. The data were compared with results derived from long-term and low dose intracerebroventricular application of the diabetogenic drug streptozotocin (STZ) known to inhibit the function of the neuronal insulin receptor and generating an insulin resistant brain state. CORT reduced both working and reference memory increasingly with time and running parallel to the STZ-induced deficit. The effect of CORT on the activities of the glycolytic enzymes hexokinase, phosphofructokinase, pyruvate kinase, glyceraldehyde-3-phosphodehydrogenase, lactate dehydrogenase and, in tricarboxylic acid cycle, α-ketoglutarate dehydrogenase equaled in both experimental conditions and in both regions studied: significant decreases of all enzyme activities except lactate dehydrogenase which increased between three and fourfold of normal. The CORT- and STZ-induced marked fall in ATP was in the same range in the regions studied. Differences became obvious in the concentration of creatine phosphate in parietotemporal cerebral cortex showing no decrease after CORT obviously due to a different susceptibility of the CORT-receptor. It is discussed that both CORT and STZ may act on the neuronal insulin receptor in a similar way. However, further studies are needed on the gene expression of insulin and the insulin receptor and its protein levels to clarify the exact action of CORT on the neuronal insulin receptor function.

Relationship between cerebral energy metabolism in parietotemporal cortex and hippocampus and mental activity during aging in rats

Summary.The present investigation demonstrates differences in both formation and utilization of the energy-rich compounds adenosine triphosphate ATP and creatine phosphate (CrP) in behaviorally well and poorly performing inbred male Wistar rats in parietotemporal cerebral cortex and hippocampus with aging from 1 to 2ys. Also, differences in learning and memory capacities (behavior) became obvious. By holeboard testing, good (GP) and poor performers (PP) have been discriminated. The pools of energy-rich phosphates as determined in parietotemporal cerebral cortex and in hippocampus under resting conditions were found to be reduced by nearly 10% for both creatine phosphate and the whole available energy pools in PP as compared to GP. Aging from 1 to 2y diminished the concentrations of energy-rich phosphates in the cerebral areas studied and in both GP and PP under resting conditions. Additionally, an age-related aggravation of the energy deficit became obvious between GP and PP. Repeated mental activation from 1y to 2y resulted in the maintenance of improvement as registered for the mean run time and the number of visited/revisited holes in GP. In contrast, PP deteriorated (mean run time), and could not maintain improvement (number of visited/revisited holes) over time. Repeated mental activation normalized the energy pool by increased formation of the energy-rich compounds ATP and CrP in both cerebral areas studied in GP and PP. However, differences became obvious between GP and PP. The energy-turnover in the latter group was found to be significantly reduced for both cerebral areas studied. GP could meet the enhanced energy demand of mental activation during aging by increasing formation and utilization of energy. PP could increase energy formation but were unable to sufficiently adapt energy utilization under the same conditions. This disturbance in energy metabolism may have impacts on energy-consuming processes in PP which may contribute to the markedly reduced cognitive reserve in PP. In human beings, PP ∼ poorly educated people found to be prone to sporadic Alzheimer disease.