This paper summarizes the proceedings of a workshop held at Trinity Hall, Cambridge to discuss comparability and includes additional information and references to related information added subsequently to the workshop. Comparability is the need to demonstrate equivalence of product after a process change; a recent publication states that this 'may be difficult for cell-based medicinal products'. Therefore a well-managed change process is required which needs access to good science and regulatory advice and developers are encouraged to seek help early. The workshop shared current thinking and best practice and allowed the definition of key research questions. The intent of this report is to summarize the key issues and the consensus reached on each of these by the expert delegates.

The development of nonviral gene delivery systems is a great challenge to enable safe gene therapy. In this study, ligand-modified nanoparticles based on human serum albumin (HSA) were developed and optimized for an efficient gene therapy. Different glutaraldehyde cross-linking degrees were investigated to optimize the HSA nanoparticles for gene delivery. The peptide sequence arginine-glycine-aspartate (RGD) and the HIV-1 transactivator of transduction sequence (Tat) are well-known as promising targeting ligands. Plasmid DNA loaded HSA nanoparticles were covalently modified on their surface with these different ligands. The transfection potential of the obtained plasmid DNA loaded RGD- and Tat-modified nanoparticles was investigated in vitro, and optimal incubation conditions for these preparations were studied. It turned out that Tat-modified HSA nanoparticles with the lowest cross-linking degree of 20% showed the highest transfection potential. Taken together, ligand-functionalized HSA nanoparticles represent promising tools for efficient and safe gene therapy.

CK2 is a serine/threonine protein kinase, which is so important for many aspects of cellular regulation that life without CK2 is impossible. Here, we analysed CK2 during adipogenic differentiation of human mesenchymal stem cells (hMSCs). With progress of the differentiation CK2 protein level and the kinase activity decreased. Whereas CK2α remained in the nucleus during differentiation, the localization of CK2β showed a dynamic shuttling in the course of differentiation. Over the last years a large number of inhibitors of CK2 kinase activity were generated with the idea to use them in cancer therapy. Our results show that two highly specific inhibitors of CK2, CX-4945 and quinalizarin, reduced its kinase activity in proliferating hMSC with a similar efficiency. CK2 inhibition by quinalizarin resulted in nearly complete inhibition of differentiation whereas, in the presence of CX-4945, differentiation proceeded similar to the controls. In this case, differentiation was accompanied by the loss of CX-4945 inhibitory function. By analysing the subcellular localization of PPARγ2, we found a shift from a nuclear localization at the beginning of differentiation to a more cytoplasmic localization in the presence of quinalizarin. Our data further show for the first time that a certain level of CK2 kinase activity is required for adipogenic stem cell differentiation and that inhibition of CK2 resulted in an altered localization of PPARγ2, an early regulator of differentiation.

Long-term high-resolution multiphoton imaging of nonlabeled human salivary gland stem cell spheroids has been performed with submicron spatial resolution, 10.5-nm spectral resolution, and picosecond temporal resolution. In particular, the two-photon-excited coenzyme NAD(P)H and flavins have been detected by time-correlated single photon counting (TCSPC). Stem cells increased their autofluorescence lifetimes and decreased their total fluorescence intensity during the adipogenic-differentiation process. In addition, the onset of the biosynthesis of lipid vacuoles was monitored over a period of several weeks in stem-cell spheroids. Time-resolved multiphoton autofluorescence imaging microscopes may become a promising tool for marker-free stem-cell characterization and cell sorting.

Gold nanoparticles are very attractive for biomedical products. However, there is a serious lack of information concerning the biological activity of nanosized gold in human tissue cells. An influence of nanoparticles on stem cells might lead to unforeseen consequences to organ and tissue functions as long as all cells arising from the initial stem cell might be subsequently damaged. Therefore the effect of negatively charged gold nanoparticles (9 and 95 nm), which are certified as reference material for preclinical biomedical research, on the adipogenic differentiation of human mesenchymal stem cells (hMSCs) is investigated here. Bone marrow hMSCs are chosen as differentiation model since bone marrow hMSCs are well characterized and their differentiation into the adipogenic lineage shows clear and easily detectable differentiation. In this study effects of gold nanoparticles on adipogenic differentiation are analyzed regarding fat storage and mitochondrial activity after different exposure times (4–21 days). Using time lapse microscopy the differentiation progress under chronically gold nanoparticle treatment is continuously investigated. In this preliminary study, chronically treatment of adipogenic differentiating hMSCs with gold nanoparticles resulted in a reduced number and size of lipid vacuoles and reduced mitochondrial activity depending on the applied concentration and the surface charge of the particles.

BackgroundDue to the genetic relationship to humans, porcine stem cells are a very important model system to investigate cell differentiation, associated cell signaling pathways, and cell fate. Porcine skin derived stem cells have been isolated from mid-gestation porcine fetus recently. To our knowledge, stem cells from the skin of the adult porcine organism have not been isolated until now. Hence, to our knowledge, we here describe the isolation, expansion, characterization and differentiation of multipotent porcine skin derived stem cell-like cells (pSSCs) from the adult porcine organism for the first time.Methodology/principal findingspSSCs had a spindle shaped morphology similar to mesenchymal stem cells (MSCs). They could be maintained proliferatively active in vitro for more than 120 days and were able to form colonies from single cells. pSSCs expressed Sox2 and Oct3/4, both transcription factors essential to the pluripotent and self-renewing phenotypes of embryonic stem cells, which recently gained attention due to their function in inducing pluripotent stem cells. Furthermore, the expression of the progenitor marker nestin, the somatic stem cell markers Bcrp1/ABCG2, Bmi1, and Stat3 was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in undifferentiated pSSCs. Flow cytometry revealed the expression of the MSC related proteins CD9, CD29, CD44 and CD105, but not CD90. After neuronal differentiation cells with a characteristic morphology of neuronal and smooth muscle-like cells were present in the cultures. Subsequent immunochemistry and flow cytometry revealed the down-regulation of nestin and the up-regulation of the neuron specific protein beta-III-tubulin and the astrocyte marker GFAP. Also, alpha-SMA expressing cells increased during differentiation suggesting the neuro-muscular differentiation of these skin derived cells. pSSCs could also be induced to differentiate into adipocyte-like cells when cultured under specific conditions.Conclusions/significanceAdult porcine skin harbors a population of stem cell-like cells (pSSCs) that can be isolated via enzymatic digestion. These pSSCs show characteristic features of MSCs originated in other tissues and express the embryonic stem cell marker Oct3/4, Sox2, and Stat3. Furthermore, pSSCs have the potential to differentiate into cells from two different germ lines, the ectoderm (neurons, astrocytes) and the mesoderm (smooth muscle cells, adipocytes).

Adult pluripotent stem cells are a cellular resource representing unprecedented potential for cell. therapy and tissue engineering. Complementary to this promise, there is a need for efficient bioprocesses for their large scale expansion and/or differentiation.With this goat in mind, our work focused on the development of three-dimensional (3-D) culture systems for controlled expansion of adult pancreatic stem cells (PSCs). For this purpose, two different culturing strategies were evaluated, using spinner vessels: cell aggregated cultures versus microcarrier technology. The use of microcarrier supports (Cytodex 1 and Cytodex 3) rendered expanded cell populations which retained their self-renewal ability, cell marker, and the potential to differentiate into adipocytes. This strategy surmounted the drawbacks of aggregates in culture which were demonstrably unfeasible as cells clumped together did not proliferate and lost PSC marker expression. Furthermore, the results obtained showed that although both microcarriers tested here were suitable for sustaining cell. expansion, Cytodex 3 provided a better substrate for the promotion of cell adherence and growth.For the latter approach, the potential of bioreactor technology was combined with the efficient Cytodex 3 strategy under controlled environmental conditions (pH-7.2, pO(2)-30% and temperature-37 degrees C); cell growth was more efficient, as shown by faster doubling time, higher growth rate and higher fold increase in cell concentration, when compared to spinner cultures. This study describes a robust bioprocess for the controlled expansion of adult PSC, representing an efficient starting point for the development of novel technologies for cell therapy. (C) 2008 Elsevier GmbH. All rights reserved.

Stem cells derived from pancreatic tissue are well characterized and exhibit a broad plasticity as they can differentiate beyond lineage boundaries into many cell types. The aim of this study was the comparative characterization of pancreatic stem cells with one other derivate of the embryonic foregut, namely salivary glands, for the existence of similar stem cell populations. The expression of stem cell markers as well as lineage-specific markers was detected by reverse transcription polymerase chain reaction, flow cytometry and immuncytochemical staining. The isolated cells from salivary glands and pancreas grew adherently in vitro and could be maintained for up to 55 and 46 population doublings, respectively. Cells from both tissues showed a comparable phenotype. They expressed different embryonic and adult stem cell markers and had the ability to differentiate spontaneously into cells representing the three embryonic germ layers. Additionally, the directed differentiation of glandular stem cells into the mesodermal lineage was achieved, yielding adipogenic, osteogenic and chondrogenic cells from salivary gland stem cells as well as osteogenic and chondrogenic cells from pancreatic stem cells. Here, we compared two stem cell populations from different glandular tissues which showed similar phenotypes and analogous properties. During embryonic development the two exocrine glands originate from the foregut, which might be the explanation for these intriguing resemblances.

Pesticides used to control unwanted insects are potentially toxic to humans. In assessing the risk involved in exposure to pesticides or complex chemical mixtures, an in vitro cell-based test can provide useful information regarding danger to human health. Cell differentiation is a biological process of fundamental importance in developing and adult organisms. In this paper, we propose a cell-based test system for continuous, label-free monitoring of the effect of test substances on stem cell differentiation. Using a prefabricated electrode-based chip and impedance measurement system, we investigated the influence of chlorpyrifos (a pesticide) on the differentiation of human mesenchymal stem cells (hMSCs) to adipocytes. The state of hMSCs on electrodes during adipogenic differentiation or after application of the cytotoxic substance was clearly reflected in the impedance measurement. Chlorpyrifos caused a partially uncovered electrode area with a decreased number of lipid vacuoles, thus leading to a rapid decrease in resistance in the cell layer. After removal of the chlorpyrifos, the cell layer resistance was regained due to the renewed covering of the electrodes by hMSCs. However, an increase in lipid vacuoles was not observed. From this, it was concluded that the measured resistance of hMSCs is determined by the electrical properties in the extra cellular space (e.g., cell/electrode or cell/cell gap), but not by the lipid vacuoles appearing in intracellular space during adipogenic differentiation.

Multiple tissue niches in the human body are now recognised to harbour stem cells. Here, we have asked how different adult stem cell populations, isolated from two ontogenetically distinct human organs (skin, pancreas), actually are with respect to a panel of standard markers/characteristics. Here we show that an easily accessible adult human tissue such as skin may serve as a convenient source of adult stem cell-like populations that share markers with stem cells derived from an internal, exocrine organ. Surprisingly, both, human pancreas- and skin-derived stem/progenitor cells demonstrate differentiation patterns across lineage boundaries into cell types of ectoderm (e.g. PGP 9.5+ and GFAP+), mesoderm (e.g. alpha-SMA+) and entoderm (e.g. amylase+ and albumin+). This intriguing differentiation capability warrants systemic follow-up, since it raises the theoretical possibility that an adult human skin-derived progenitor cell population could be envisioned for possible application in cell replacement therapies.

Detection of differentiation in general and adipogenesis specifically is conventionally practised by taking only the few cells into account which are visible in the field of view provided by optical microscopy using high-resolution objectives. Other methods of quantification of adipogenic differentiation such as real time PCR, measurement of glycero phosphate dehydrogenase activity or adipogenesis assays only provide integral information tacking spatial resolution and information on the fraction of differentiated cells.Here we used high-resolution scanning and automated image processing to automatically analyze and quantify cell numbers in the range of 20,000. For optimisation of the approach, human gland stem cells (GSC) were differentiated to the adipogenic phenotype comprising inclusion of lipid vesicles. Oil. red 0 and 4', 6'-diamidino-2-phenylindole (DAPI) staining made it possible to derive the number of differentiated cells in relation to the total number of cells. For evaluation of the image processing software we verified our results using adipogenesis assay and phase contrast based cell counting.We developed a method of determining differentiation efficiencies covering the range from 10% down to 100ppm with the same image processing and an identical set of parameters, matching the results of the adipogenesis assay. Our approach is based on a statistically significant number of cells and shows high sensitivity taking into account the heterogeneous differentiation pattern of adipogenesis in GSC and other stem cells. (C) 2008 Elsevier GmbH. All rights reserved.

A variety of human and animal stem cells (rat and human adult pancreatic stem cells, salivary gland stem cells, dental pulpa stem cells) have been investigated by femtosecond laser 5D two-photon microscopy. Autofluorescence and second harmonic generation have been imaged with submicron spatial resolution, 270 ps temporal resolution, and 10 nm spectral resolution. In particular, NADH and flavoprotein fluorescence was detected in stem cells. Major emission peaks at 460mn and 530nm with typical mean fluorescence lifetimes of 1.8 ns and 2.0 ns, respectively, were measured using time-correlated single photon counting and spectral imaging. Differentiated stem cells produced the extracellular matrix protein collagen which was detected by SHG signals at 435 mn.

Novel ultracompact multiphoton sub-20 femtosecond near infrared 85 MHz laser scanning microscopes and conventional 250 fs laser microscopes have been used to perform high spatial resolution two-photon imaging of stem cell clusters as well as selective intracellular nanoprocessing and knock out of living single stem cells within an 3D microenvironment without any collateral damage. Also lethal cell exposure of large parts of cell clusters was successfully probed while maintaining single cells of interest alive. The mean power could be kept in the milliwatt range for 3D nanoprocessing and even in the microwatt range for two-photon imaging. Ultracompact low power sub-20 fs laser systems may become interesting tools for optical nanobiotechnology such as optical cleaning of stem cell clusters as well as optical transfection.

Adult human and rat pancreas stem cells as well as tumor spheroids were irradiated with femtosecond laser pulses in the near infrared (NIR) spectral range at high transient GW/cm(2) and TW/cm(2) intensities. The cellular response to the laser exposure was probed by the detection of modifications of NAD(P)H autofluorescence, the formation of reactive oxygen species (ROS) and DNA strand breaks (TUNEL-assay), and viability (live/dead test). The results confirm that long-term scanning of stem cells can be performed at appropriate laser exposure parameters without a measurable impact on the cellular metabolism and vitality. In addition, it was proven that a targeted inactivation of a particular single stem cells or a single tumour cell inside a 3D cell cluster using single point illumination at TW/cm(2) laser intensities can be performed without affecting neighbouring cells.Therefore multiphoton microscopes can be considered as biosafe tools for long-term analysis of stem cells as well as highly precise optical knocking out of single cells within cell clusters and tissues.

Adult stem cells in general have the potential to differentiate into different cell types. Bone marrow, in particular, contains a mesenchymal progenitor cell that can give rise to many other cell types, such as osteocytes, adipocytes, muscle cells, astrocytes, and neurons, as well as stromal cells that support hematopoiesis. In postnatal organisms, each tissue and organ is now generally perceived to contain a small sub-population of quiescent cells, which are, when stimulated accordingly, capable of self-renewal and of indefinite or at least prolonged proliferative potential. Regenerative medicine tries to exploit this fact by directing differentiation to specific cell types that can subsequently be transplanted to replace damaged tissue. Among the many challenges that need to be faced with regard to future exploitation, observation of the differentiation process and determination of the differentiation state of the cells without harming them are of prominent importance. We employed time-resolved acoustic microscopy in adult stem cell differentiation for monitoring the differentiation process and characterizing the degree of differentiation of individual cells.