In vitro-differentiated T/natural killer-cell progenitors derived from human CD34+ cells mature in the thymus.

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is a treatment option for patients with hematopoietic malignancies that is hampered by treatment-related morbidity and mortality, in part the result of opportunistic infections, a direct consequence of delayed T-cell recovery. Thymic output can be improved by facilitation of thymic immigration, known to require precommitment of CD34(+) cells. We demonstrate that Delta-like ligand-mediated predifferentiation of mobilized CD34(+) cells in vitro results in a population of thymocyte-like cells arrested at a T/natural killer (NK)-cell progenitor stage. On intrahepatic transfer to Rag2(-/-)gamma(c)(-/-) mice, these cells selectively home to the thymus and differentiate toward surface T-cell receptor-alphabeta(+) mature T cells considerably faster than animals transplanted with noncultured CD34(+) cells. This finding creates the opportunity to develop an early T-cell reconstitution therapy to combine with HSCT.

T cells fail to develop in the human skin-cell explants system; an inconvenient truth.

BACKGROUND: Haplo-identical hematopoietic stem cell (HSC) transplantation is very successful in eradicating haematological tumours, but the long post-transplant T-lymphopenic phase is responsible for high morbidity and mortality rates. Clark et al. have described a skin-explant system capable of producing host-tolerant donor-HSC derived T-cells. Because this T-cell production platform has the potential to replenish the T-cell levels following transplantation, we set out to validate the skin-explant system. RESULTS: Following the published procedures, while using the same commercial components, it was impossible to reproduce the skin-explant conditions required for HSC differentiation towards mature T-cells. The keratinocyte maturation procedure resulted in fragile cells with minimum expression of delta-like ligand (DLL). In most experiments the generated cells failed to adhere to carriers or were quickly outcompeted by fibroblasts. Consequently it was not possible to reproduce cell-culture conditions required for HSC differentiation into functional T-cells. Using cell-lines over-expressing DLL, we showed that the antibodies used by Clark et al. were unable to detect native DLL, but instead stained 7AAD+ cells. Therefore, it is unlikely that the observed T-lineage commitment from HSC is mediated by DLL expressed on keratinocytes. In addition, we did confirm expression of the Notch-ligand Jagged-1 by keratinocytes. CONCLUSIONS: Currently, and unfortunately, it remains difficult to explain the development or growth of T-cells described by Clark et al., but for the fate of patients suffering from lymphopenia it is essential to both reproduce and understand how these co-cultures really "work". Fortunately, alternative procedures to speed-up T-cell reconstitution are being established and validated and may become available for patients in the near future.

Lysosomal dysfunction in muscle with special reference to glycogen storage disease type II.

The importance of proper lysosomal activity in cell and tissue homeostasis is underlined by "experiments of nature", i.e. genetic defects in one of the at least 40 lysosomal enzymes/proteins present in the human cell. The complete lack of 1-4 alpha-glucosidase (glycogen storage disease type II (GSD II) or Pompe disease) is life-threatening. Patients suffering from GSD II commonly die before the age of 2 years because of cardiorespiratory insufficiency. Striated muscle cells appear to be particularly vulnerable in GSD II. The high cytoplasmic glycogen content in muscle cells most likely gives rise to a high rate of glycogen engulfment by the lysosomes. The polysaccharides become subsequently trapped in these organelles when 1-4 alpha-glucosidase activity is absent. During the course of the disease, muscle wasting occurs. It is hypothesised that the gradual loss of muscle mass is caused by a combination of disuse atrophy and lipofuscine-mediated apoptosis of myocytes. Moreover, we hypothesise that in the remaining skeletal muscle cells, longitudinal transmission of force is hampered by swollen lysosomes, clustering of non-contractile material and focal regions with degraded contractile proteins, which results in muscle weakness.

Effects of non-contractile inclusions on mechanical performance of skeletal muscle.

Glycogen storage disease II is an inherited progressive muscular disease in which the lack of functional acid 1-4 alpha-glucosidase results in the accumulation of lysosomal glycogen. In the present study, we examine the effect of these non-contractile inclusions on the mechanical performance of skeletal muscle. To this end, force developed in an isometrically contracting slice of a muscle was calculated with a finite element model. Force was calculated at several inclusion densities and distributions and compared to muscle lacking inclusions. Furthermore, ankle dorsal flexor torque was measured in situ of alpha-glucosidase null mice of 6 months of age and unaffected litter mates as was inclusion density in the dorsal flexor muscles. The calculated force loss was shown to be almost exclusively dependent on the inclusion density and less on the type of inclusion distribution. The force loss predicted by the model (6%) on the basis of measured inclusion density (3.3%) corresponded to the loss in mass-normalized strength in these mice measured in situ (7%). Therefore, we conclude that the mechanical interaction between the non-contractile inclusions and the nearby myofibrils is a key factor in the loss of force per unit muscle mass during early stages of GSD II in mice. As glycogen accumulation reaches higher levels in humans, it is highly probable that the impact of this mechanical interaction is even more severe in human skeletal muscle.

Age-related morphological changes in skeletal muscle cells of acid alpha-glucosidase knockout mice.

Glycogen storage disease type II (GSDII), caused by a genetic defect in acid alpha-glucosidase (AGLU), leads to a decline in muscle contractility caused by both muscle wasting and a decrease in muscle quality, i.e., force generated per unit muscle mass. A previous study has shown that loss of muscle mass can only explain one-third of the decrease in contractile performance. Here we report on changes in the intramyocellular structural organization in a mouse knockout model (AGLU(-/-) mice) as a possible cause for the decline in muscle quality. Swollen, glycogen-filled lysosomes and centrally localized cores with cellular debris partially contribute to the decline in muscle quality. Altered localization and deposition of cytoskeletal proteins desmin and titin may reflect adaptive mechanisms at the age of 13 months, but a decline in quality at 20 months of age. The early deposition of lipofuscin in AGLU-deficient myocytes (13 months) is most likely a reflection of enhanced oxidative stress, which may also affect muscle quality. These collective findings, on the one hand, may explain the decrease in tissue quality and, on the other, may represent markers for efficacy of therapeutic interventions to restore muscle function in patients suffering from GSDII.

Age-related decline in muscle strength and power output in acid 1-4 alpha-glucosidase knockout mice.

A hallmark of glycogen storage disease type II, caused by defective alpha-glucosidase (AGLU) activity, is a progressive decline in muscle performance. The objective of this study was to determine the relative contribution to this decline in muscle performance of (1) decline in muscle mass; (2) decline in muscle protein content per unit mass; and (3) accumulation of glycogen. To this end, isometric torque and power in AGLU(-/-) mice at 7, 13, and 20 months were assessed in situ. Power (approximately 24 mW) and torque (approximately 2.45 Nmm) did not change with age in control animals, but declined significantly in AGLU(-/-) mice, in the three age groups. No decline in protein content per unit muscle mass was observed. Muscle atrophy explained one third of the decline in muscle performance; the remaining part was attributed to a decrease in muscle quality--a decrease in mechanical performance per unit muscle mass. Mechanical effects of glycogen inclusions could not fully explain this decrease. Additional factors must therefore play a role.

A modified PAS stain combined with immunofluorescence for quantitative analyses of glycogen in muscle sections.

Simultaneous analyses of glycogen in sections with other subcellular constituents within the same section will provide detailed information on glycogen deposition and the processes involved. To date, staining protocols for quantitative glycogen analyses together with immunofluorescence in the same section are lacking. We aimed to: (1) optimise PAS staining for combination with immunofluorescence, (2) perform quantitative glycogen analyses in tissue sections, (3) evaluate the effect of section thickness on PAS-derived data and (4) examine if semiquantitative glycogen data were convertible to genuine glycogen values. Conventional PAS was successfully modified for combined use with immunofluorescence. Transmitted light microscopic examination of glycogen was successfully followed by semiquantification of glycogen using microdensitometry. Semiquantitative data correlated perfectly with glycogen content measured biochemically in the same sample (r2=0.993, P<0.001). Using a calibration curve (r2=0.945, P<0.001) derived from a custom-made external standard with incremental glycogen content, we converted the semiquantitative data to genuine glycogen values. The converted semiquantitative data were comparable with the glycogen values assessed biochemically (P=0.786). In addition we showed that for valid comparison of glycogen content between sections, thickness should remain constant. In conclusion, the novel protocol permits the combined use of PAS with immunofluorescence and shows valid conversion of data obtained by microdensitometry to genuine glycogen data.

Impaired performance of skeletal muscle in alpha-glucosidase knockout mice.

Glycogen storage disease type II (GSD II) is an inherited progressive muscle disease in which lack of functional acid alpha-glucosidase (AGLU) results in lysosomal accumulation of glycogen. We report on the impact of a null mutation of the acid alpha-glucosidase gene (AGLU(-/-)) in mice on the force production capabilities, contractile mass, oxidative capacity, energy status, morphology, and desmin content of skeletal muscle. Muscle function was assessed in halothane-anesthetized animals, using a recently designed murine isometric dynamometer. Maximal torque production during single tetanic contraction was 50% lower in the knockout mice than in wild type. Loss of developed torque was found to be disproportionate to the 20% loss in muscle mass. During a series of supramaximal contraction, fatigue, expressed as percentile decline of developed torque, did not differ between AGLU(-/-) mice and age-matched controls. Muscle oxidative capacity, energy status, and protein content (normalized to either dry or wet weight) were not changed in knockout mice compared to control. Alterations in muscle cell morphology were clearly visible. Desmin content was increased, whereas alpha-actinin was not. As the decline in muscle mass is insufficient to explain the degree in decline of mechanical performance, we hypothesize that the large clusters of noncontractile material present in the cytoplasm hamper longitudinal force transmission, and hence muscle contractile function. The increase in muscular desmin content is most likely reflecting adaptations to altered intracellular force transmission.