Margriet Huisman

Stem cells from the adult hair follicle bulge can differentiate into neurons and glia, which is advantageous for the development of an autologous cell-based therapy for neurological diseases. Consequently, bulge stem cells from plucked hair may increase opportunities for personalized neuroregenerative therapy. Hairs were plucked from the scalps of healthy donors, and the bulges were cultured without prior tissue treatment. Shortly after outgrowth from the bulge, cellular protein expression was established immunohistochemically. The doubling time was calculated upon expansion, and the viability of expanded, cryopreserved cells was assessed after shear stress. The neuroglial differentiation potential was assessed from cryopreserved cells. Shortly after outgrowth, the cells were immunopositive for nestin, SLUG, AP-2α and SOX9, and negative for SOX10. Each bulge yielded approximately 1 × 10(4) cells after three passages. Doubling time was 3.3 (±1.5) days. Cellular viability did not diff...

In severely anemic fetuses of women alloimmunized to RBC antigens, transfused donor RBCs disappear faster than in adults. This may result from an accelerated linear or nonlinear decline with time. It was investigated whether changes in donor RBC age characteristics after circulation in the fetus may reflect the main type of cellular decline. Donor RBC age characteristics (density, mean cell volume [MCV], and mean cell Hb content [MCHC]) were determined before intrauterine transfusions. Density gradient centrifugation was used to obtain RBCs of different ages. The results from gradient centrifugation were used to calculate mean values for the density, MCV, and MCHC to be expected after the transfusion interval, assuming a linear decline in RBCs of 1 percent per day. Donor and fetal RBCs, taken just before the second transfusion, were separated by agglutination with IgM D MoAb. For these donor cells, the observed mean values for density, MCV, and MCHC were compared with the calculated, expected values (n = 12). The mean +/- SD transfusion interval was 17.9 +/- 3.6 days. The Hb declined by 1.75 +/- 0.62 percent per day (n = 9). After the transfusion interval and contrary to the expected changes, cell density and MCHC decreased and MCV increased significantly (0. 001<p<0.02). This difference between actual and calculated values decreased with increasing intervals; for MCV, it was also associated with a greater decline in Hb per day (p<0.05). All donor cells age during circulation in the fetus. However, after the transfusion interval, the donor RBC population remaining is apparently younger than the RBC population before transfusion. This results from a preferential disappearance of older donor RBCs and not from a linear loss of cells with time. The removal of older RBCs before the transfusion may increase the time between transfusions and thereby reduce the total number of transfusions required.

Background Hearing depends on correct functioning of the cochlear hair cells, and their innervation by spiral ganglion neurons. Most of the insight into the embryological and molecular development of this sensory system has been derived from animal studies. In contrast, little is known about the molecular expression patterns and dynamics of signaling molecules during normal fetal development of the human cochlea. In this study, we investigated the onset of hair cell differentiation and innervation in the human fetal cochlea at various stages of development. Results At 10 weeks of gestation, we observed a prosensory domain expressing SOX2 and SOX9/SOX10 within the cochlear duct epithelium. In this domain, hair cell differentiation was consistently present from 12 weeks, coinciding with downregulation of SOX9/SOX10, to be followed several weeks later by downregulation of SOX2. Outgrowing neurites from spiral ganglion neurons were found penetrating into the cochlear duct epithelium pri...

Round window membrane (RWM) application of ouabain is known to selectively destroy type I spiral ganglion cells (SGCs) in cochleas of several rodent species, while leaving hair cells intact. This protocol has been used in rats and Mongolian gerbils, but observations in the guinea pig are conflicting. This is why we reinvestigated the effect of ouabain on the guinea pig cochlea. Ouabain solutions of different concentrations were placed, in a piece of gelfoam, upon the RWM of the right cochleas. Auditory function was assessed using acoustically evoked auditory brainstem responses (aABR). Finally, cochleas were fixed and processed for histological examination. Due to variability within treatment groups, histological data was pooled and three categories based upon general histological observations were defined: cochleas without outer hair cell (OHC) and SGC loss (Category 1), cochleas with OHC loss only (Category 2), and cochleas with OHC and SGC loss (Category 3). Animals treated with ...

To investigate differences in cell proliferation, cell cycle arrest and apoptosis between cholesteatoma and control skin. Immunohistochemical sections of 15 cholesteatoma and 15 paired control retro-auricular skin samples were examined for Ki-67, p53, p21 and active caspase 3, using image analysis, as well as for DNA fragmentation. The retro-auricular skin samples contained 5.7% +/- 3.6%, Ki-67-positive cells and showed a normal expression pattern. In the cholesteatoma epithelium 11.7% +/- 9.5% of the cells were Ki-67-positive and these cells were dominantly expressed in the basal and parabasal cell layers. Retro-auricular skin contained 5.8% +/- 5.4% p53-positive cells and 1.0% +/- 0.9%, p21-positive cells. In the cholesteatoma epithelium 17.8% +/- 12.3% of the cells were p53-positive and 14.3% +/- 11.6% were p21-positive The expression of Ki-67, p53 and p21 differed significantly between the two groups (p < 0.05). In the cholesteatoma epithelium a positive correlation was found between p53 and p21 expression (p = 0.016). Active caspase 3 positivity and DNA fragmentation were rarely seen in the cholesteatoma epithelium. Our results indicate that increased cell proliferation in cholesteatoma epithelium is accompanied by an increase in p53 and p21 protein levels, whilst apoptosis is minimal.

Class III β-tubulin (TUBB3)-positive cells from the hair follicle bulge are thought to be neuronal cells derived from a local neural crest stem cell. However, TUBB3 has recently been shown to be expressed in the melanocytic lineage. To evaluate the neural-crest-associated immunophenotype of TUBB3-positive cells from hair follicle bulge explants, we dissected hair follicle bulges out from mouse whisker pads and cultured for 1 month and assessed outgrowing cells by means of immunocytochemistry using the biomarkers TUBB3, nestin, NGFR, SOX9, TYRP1 and laminin. Large amounts of TUBB3-positive cells could be cultured that co-expressed nestin, NGFR, SOX9 and, to a lesser degree, TYRP1, matching a melanoglial phenotype. In addition, a small population of TUBB3-negative but laminin-positive cells was found, which presumably are of glial origin. It can be concluded that cells of melanoglial origin can easily be obtained from hair follicle bulge explants. These cells may be of use in experime...

Sensorineural hearing loss (SNHL) is one of the most common congenital disorders in humans, afflicting one in every thousand newborns. The majority is of heritable origin and can be divided in syndromic and nonsyndromic forms. Knowledge of the expression profile of affected genes in the human fetal cochlea is limited, and as many of the gene mutations causing SNHL likely affect the stria vascularis or cochlear potassium homeostasis (both essential to hearing), a better insight into the embryological development of this organ is needed to understand SNHL etiologies. We present an investigation on the development of the stria vascularis in the human fetal cochlea between 9 and 18 weeks of gestation (W9-W18) and show the cochlear expression dynamics of key potassium-regulating proteins. At W12, MITF+/SOX10+/KIT+ neural-crest-derived melanocytes migrated into the cochlea and penetrated the basement membrane of the lateral wall epithelium, developing into the intermediate cells of the st...

There is a strong indication that epithelial keratinocytes in cholesteatoma are protected against apoptosis. The late terminal differentiation program in cholesteatoma epithelium is disturbed. Previously, minimal apoptosis has been demonstrated in cholesteatoma epithelium. The phosphoinositide 3-kinase/Akt/protein kinase B (PI3K/Akt/PKB) and the mitogen activated protein kinases (MAPK) signaling transduction pathways have been reported to protect epithelial cells against apoptosis. Both pathways have also been proven to regulate late terminal differentiation of keratinocytes. In cholesteatoma epithelium, MAPK activation has been shown to be associated with terminal differentiation. The purpose of this study was to investigate whether in human cholesteatoma epithelium protection against programmed cell death by means of PI3K/Akt survival signaling is present and associated with MAPK activation and terminal differentiation. Fifteen human cholesteatoma and patient-matched retro-auricular skin samples were immunohistochemically stained for pAkt/PKB, phosphorylated extracellular regulated kinase1/2 (pERK1/2), phosphorylated JNK/SAPK, phosphorylated p38, involucrin and filaggrin. Positive cells were counted by computer-assisted digital image analysis. Protein expressions of pAkt/PKB, pERK1/2, pp38, and involucrin in cholesteatoma epithelium were significantly increased when compared with retro-auricular skin (p<0.01). Filaggrin expression was significantly decreased (p=0.03). The positive correlation was confirmed between both pERK1/2 and pp38, and involucrin (p < or = 0.05).