Vom Wissenschaftler zum Unternehmer – eine strategische Herausforderung

info:eu-repo/semantics/publishe

Development of an <i>in vitro</i> potency assay for human skeletal muscle derived cells

<div><p>Background</p><p>Potency is a quantitative measure of the desired biological function of an advanced therapy medicinal product (ATMP) and is a prerequisite for market approval application (MAA). To assess the potency of human skeletal muscle-derived cells (SMDCs), which are currently investigated in clinical trials for the regeneration of skeletal muscle defects, we evaluated acetylcholinesterase (AChE), which is expressed in skeletal muscle and nervous tissue of all mammals.</p><p>Methods</p><p>CD56⁺ SMDCs were separated from CD56^- SMDCs by magnetic activated cell sorting (MACS) and both differentiated in skeletal muscle differentiation medium. AChE activity of <i>in vitro</i> differentiated SMDCs was correlated with CD56 expression, fusion index, cell number, cell doubling numbers, differentiation markers and compared to the clinical efficacy in patients treated with SMDCs against fecal incontinence.</p><p>Results</p><p>CD56^- SMDCs did not form multinucleated myotubes and remained low in AChE activity during differentiation. CD56⁺ SMDCs generated myotubes and increased in AChE activity during differentiation. AChE activity was found to accurately reflect the number of CD56⁺ SMDCs in culture, their fusion competence, and cell doubling number. In patients with fecal incontinence responding to SMDCs treatment, the improvement of clinical symptoms was positively linked with the AChE activity of the SMDCs injected.</p><p>Discussion</p><p>AChE activity was found to truly reflect the <i>in vitro</i> differentiation status of SMDCs and to be superior to the mere use of surface markers as it reflects not only the number of myogenic SMDCs in culture but also their fusion competence and population doubling number, thus combining cell quality and quantification of the expected mode of action (MoA) of SMDCs. Moreover, the successful <i>in vitro</i> validation of the assay proves its suitability for routine use. Most convincingly, our results demonstrate a link between clinical efficacy and the AChE activity of the SMDCs preparations used for the treatment of fecal incontinence. Thus, we recommend using AChE activity of <i>in vitro</i> differentiated SMDCs as a potency measure in end stage (phase III) clinical trials using SMDCs for skeletal muscle regeneration and subsequent market approval application (MAA).</p></div

Molecular determinants of inactivation in voltage-gated Ca2+ channels

Evolution has created a large family of different classes of voltage-gated Ca2+ channels and a variety of additional splice variants with different inactivation properties. Inactivation controls the amount of Ca2+ entry during an action potential and is, therefore, believed to play an important role in tissue-specific Ca2+ signalling. Furthermore, mutations in a neuronal Ca2+ channel (Cav2.1) that are associated with the aetiology of neurological disorders such as familial hemiplegic migraine and ataxia cause significant changes in the process of channel inactivation. Ca2+ channels of a given subtype may inactivate by three different conformational changes: a fast and a slow voltage-dependent inactivation process and in some channel types by an additional Ca2+-dependent inactivation mechanism. Inactivation kinetics of Ca2+ channels are determined by the intrinsic properties of their pore-forming α1-subunits and by interactions with other channel subunits. This review focuses on structural determinants of Ca2+ channel inactivation in different parts of Ca2+ channel α1-subunits, including pore-forming transmembrane segments and loops, intracellular domain linkers and the carboxyl terminus. Inactivation is also affected by the interaction of the α1-subunits with auxiliary β-subunits and intracellular regulator proteins. The evidence shows that pore-forming S6 segments and conformational changes in extra- (pore loop) and intracellular linkers connected to pore-forming segments may play a principal role in the modulation of Ca2+ channel inactivation. Structural concepts of Ca2+ channel inactivation are discussed

Comparison of AChE activity with CD56 expression and fusion competence.

<p>AChE activity per g of total protein was determined in SMDC populations derived from at least three different patients with different percentages of CD56⁺ cells after 6 days of differentiation on gelatin coated 24-well plates. Correlation between AChE and CD56 expression in SMDCs by Pearson linear regression analysis of at least 4 different patient samples results at a <i>p</i>-value of <i>p</i> = 0.0096, a correlation coefficient of <i>r</i> = 0.9599 and coefficient of determination of <i>r</i>^<i>2</i> = 0.9214. Data points were presented as mean±SEM AChE activity. 95% confidence intervals are shown as dotted lines (A). Correlation between AChE and fusion index (FI) of SMDCs by Pearson linear regression analysis of at least 4 different patient samples resulted in a <i>p</i>-value of <i>p</i> = 0.0266, a correlation coefficient of <i>r</i> = 0.9734 and coefficient of determination of <i>r</i>^<i>2</i> = 0.9475. Data points presented as mean±SEM AChE activity and mean±SEM fusion index of four different patient samples in a total of 16 mixtures of fusion competent and non-fusogenic cells. 95% confidence intervals are shown as dotted lines (B). SMDCs from 4 patients resulting in a total of 16 SMDCs populations grouped in “High AChE”, “Medium AChE”, “Low AChE” and “Very low AChE” according to their mean±SEM AChE activity of 4421±540, 1774±176, 1302±179 and 398±27 AChEmUrel/g protein, respectively stained for SK-Myosin expression after 6 days of differentiation on gelatin-coated 24-well plates. Scale bar = 100 μm (C).</p

Raw data figure 1.xlsx

Raw data of Figure 1 in<div><br></div><div>doi:10.1371/journal.pone.0194561<div><br></div><div><br></div></div

The central role of AChE as potency measure for SMDCs.

<p>SMDC quality attributes such as purity of CD56⁺ cells and population doubling numbers are connected to potency by AChE activity, which reflects the expected mode of action of SMDCs, is linked to clinical efficacy and might be useful in effective dose definition.</p

AChE activity and clinical efficacy.

<p>Percent reduction of IEF from baseline to 6 months in responders receiving Low (n = 44), Medium (n = 15) or High (n = 11) and non-responders also receiving Low (n = 48), Medium (n = 22) and High (n = 9) total AChE activity SMDC batches (A). Percentage of CD56 positive cells (B), total number of cells (C), and AChE activity [mU_rel] per 2*10⁵ (200T) cells (D) in all SMDCs batches used for treatment of fecal incontinence in responder and non-responder patients according to total AChE activity. Data presented as mean and 95% confidence interval. Groups were compared by two-tailed unpaired <i>t</i>-test with Welch’s correction. A <i>p</i>-value below 0.05 was considered as significant (*) n.s.: not-significant.</p

SMDC isolation, differentiation and AChE quantification.

<p>CD56⁺ and CD56^- cells were separated by MACS. Successful separation was confirmed by flow cytometry. Positively selected cells from five different human muscle biopsies using anti CD56 MACS antibody contain 96.93 ± 1.64% CD56⁺ cells (representative histogram in purple) whereas negatively selected cells from five individual patients contain 4.25 ± 3.89% CD56⁺ cells (representative histogram in green). White histograms represent isotype control staining (A). When cultured in skeletal muscle differentiation medium, single nucleated CD56⁺ SMDCs fuse and produce multinucleated tubes on gelatin coated 24-well plates with a fusion index (FI) of 67.90 ± 11.06 (mean ± SD; n = 3) after 6 days of differentiation (B). CD56^- SMDCs do not form myotubes within 6 days of cultivation in differentiation medium on gelatin coated 24-well plates (FI = 1.26 ± 2.16, n = 3) (C). The change in AChE activity per g total protein of 200 000 CD56⁺ cells seeded on gelatin-coated 24-well plates before induction of differentiation (Day 0) and 6 days after differentiation (Day 6) is significantly different (p<0.05) in a ratio paired <i>t</i>-test of at least three patient samples (data presented as mean ± SEM) (D) but not significantly (n.s.) different between AChE activity in three batches of CD56^- cells before and 6 days after induction of skeletal muscle differentiation in a ratio paired <i>t</i>-test of three different SMDC patient samples (E). Scale bar (white) = 100 μm.</p