An in vitro limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse

Abstract We have developed a limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse using a miniturized stroma- dependent bone marrow culture assay in vitro. The cells were overlaid on irradiated stromal layers in microtiter wells in a range of concentrations, and frequencies of cobblestone area-forming cells (CAFC) were calculated by employing Poisson statistics. The production of secondary granulocyte/macrophage colony-forming units (CFU-G/M) in the adherent layer of individual wells was correlated with the presence of such cobblestone areas. CAFC frequencies were determined in bone marrow cell suspensions that were either enriched for marrow repopulating ability (MRA) in vivo, while depleted for spleen colony- forming units (CFU-S), or vice versa. The separation of bone marrow cells (BMC) was either based on centrifugal elutriation, or monoclonal antibody-mediated magnetic depletion of cells carrying cell surface differentiation antigens, and subsequent sorting on the basis of light scatter and rhodamine-123 retention as a measure of mitochondrial activity. In addition, 5-fluorouracil-resistant BMC were studied. Our investigations show that a time-dependent cobblestone area formation exists that reflects the turnover time and primitiveness of CAFC. The frequency of precursors forming cobblestone areas on day 28 after overlay is proposed to be a measure for MRA, whereas the day-7 CAFC frequency closely corresponds with day-12 CFU-S numbers in the suspensions tested.</jats:p

An in vitro limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse

We have developed a limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse using a miniturized stroma- dependent bone marrow culture assay in vitro. The cells were overlaid on irradiated stromal layers in microtiter wells in a range of concentrations, and frequencies of cobblestone area-forming cells (CAFC) were calculated by employing Poisson statistics. The production of secondary granulocyte/macrophage colony-forming units (CFU-G/M) in the adherent layer of individual wells was correlated with the presence of such cobblestone areas. CAFC frequencies were determined in bone marrow cell suspensions that were either enriched for marrow repopulating ability (MRA) in vivo, while depleted for spleen colony- forming units (CFU-S), or vice versa. The separation of bone marrow cells (BMC) was either based on centrifugal elutriation, or monoclonal antibody-mediated magnetic depletion of cells carrying cell surface differentiation antigens, and subsequent sorting on the basis of light scatter and rhodamine-123 retention as a measure of mitochondrial activity. In addition, 5-fluorouracil-resistant BMC were studied. Our investigations show that a time-dependent cobblestone area formation exists that reflects the turnover time and primitiveness of CAFC. The frequency of precursors forming cobblestone areas on day 28 after overlay is proposed to be a measure for MRA, whereas the day-7 CAFC frequency closely corresponds with day-12 CFU-S numbers in the suspensions tested.</jats:p

Functional hindrance due to spasticity in individuals with spinal cord injury during inpatient rehabilitation and 1 year thereafter

Study design: Prospective cohort study. Objective: To assess functional hindrance due to spasticity during inpatient rehabilitation and 1 year thereafter in individuals with spinal cord injury (SCI) and to determine factors that influence the hindrance. Setting: Eight specialized rehabilitation centres in the Netherlands. Methods: A total of 203 patients with recent SCI rated the hindrance they perceived due to spasticity in daily living at the start of active rehabilitation (t1), 3 months later (t2), at discharge (t3) and 1 year after discharge (t4). Hindrance was dichotomized into absent or negligible and present. Multilevel regression analyses were performed to determine the course of functional hindrance due to spasticity and its associations with possible determinants-namely, age, gender, cause, lesion level, motor completeness, spasticity and anti-spasticity medication. Results: The percentage of individuals that indicated functional hindrance due to spasticity ranged from 54 to 62% over time and did not change significantly over time (Delta t3t1 odds ratio (OR)=0.85, P=0.44;Delta t3t2 OR=1.20, P=0.41;Delta t3t4 OR=0.91, P=0.67). The percentage of individuals who experienced a lot of hindrance due to spasticity during specific activities ranged from 4 to 27%. The odds for experiencing functional hindrance due to spasticity were significantly higher for individuals with tetraplegia (OR=2.17, P=0.0001), more severe spasticity (OR=5.51, P Conclusion: Functional hindrance due to spasticity occurred in the majority of persons with SCI and did not change significantly during inpatient rehabilitation and 1 year thereafter. Factors that influence hindrance were determined

Perception of lower limb spasticity in patients with spinal cord injury

Patients with complete SCI experienced several manifestations of spasticity, extensor spasms being the most common. Many daily life activities elicited different manifestations of spasticity. The experienced discomfort was only moderately related to the perceived degree of spasticity during an activity. Possibly, the discomfort is influenced by other factors than the perceived spasticity alone.\u