Identification of cardiac stem cells within mature cardiac myocytes

Cardiac stem cells are described in a number of mammalian species including humans. Cardiac stem cell clusters consisting of both lineage-negative and partially committed cells are generally identified between contracting cardiac myocytes. In the present study, c-kit⁺, Sca⁺, and Isl1⁺ stem cells were revealed to be located inside the sarcoplasm of cardiac myocytes in myocardial cell cultures derived from newborn, 20-, and 40-day-old rats. Intracellularly localized cardiac stem cells had a coating or capsule with a few pores that opened into the host cell sarcoplasm. The similar structures were also identified in the suspension of freshly isolated myocardial cells (ex vivo) of 20- and 40-day-old rats. The results from this study provide direct evidence for the replicative division of encapsulated stem cells, followed by their partial cardiomyogenic differentiation. The latter is substantiated by the release of multiple transient amplifying cells following the capsule rupture. In conclusion, functional cardiac stem cells can reside not only exterior to but also within cardiomyocytes.     

Human cell lines for biopharmaceutical manufacturing: history,status, and future perspectives

Biotherapeutic proteins represent a mainstay of treatment for a multitude of conditions, for example, autoimmune disorders, hematologic disorders, hormonal dysregulation, cancers, infectious diseases and genetic disorders. The technologies behind their production have changed substantially since biotherapeutic proteins were first approved in the 1980s. Although most biotherapeutic proteins developed to date have been produced using the mammalian Chinese hamster ovary and murine myeloma (NS0, Sp2/0) cell lines, there has been a recent shift toward the use of human cell lines. One of the most important advantages of using human cell lines for protein production is the greater likelihood that the resulting recombinant protein will bear post-translational modifications (PTMs) that are consistent with those seen on endogenous human proteins. Although other mammalian cell lines can produce PTMs similar to human cells, they also produce non-human PTMs, such as galactose-α1,3-galactose and N-glycolylneuraminic acid, which are potentially immunogenic. In addition, human cell lines are grown easily in a serum-free suspension culture, reproduce rapidly and have efficient protein production. A possible disadvantage of using human cell lines is the potential for human-specific viral contamination, although this risk can be mitigated with multiple viral inactivation or clearance steps. In addition, while human cell lines are currently widely used for biopharmaceutical research, vaccine production and production of some licensed protein therapeutics, there is a relative paucity of clinical experience with human cell lines because they have only recently begun to be used for the manufacture of proteins (compared with other types of cell lines). With additional research investment, human cell lines may be further optimized for routine commercial production of a broader range of biotherapeutic proteins.     

In-vitro formation of a collagenous matrix upon previously diseased and experimentally treated cemental root surfaces

Summary A diseased and mechanically treated surface of root cementum is known, clinically, to favor periodontal regeneration. The present investigation was undertaken to test whether previously diseased and experimentally treated root surfaces can support the in-vitro formation of a new collagenous matrix. Three teeth extracted for advanced periodontitis were treated first with 5% sodium hypochlorite for 2 h to remove all organic material from the root surface. After the healthy, apical one third of the root was cut off, the roots were scaled with moderate pressure to remove visible calculus. Non-demineralized root discs were cut and placed on a co-culture of periodontal ligament- and alveolar bone-derived cells. After 7 weeks in culture, either one of two matrix types was found along the root surface. The most frequent matrix consisted of clusters of cells layered within densely aggregated collagen fibrils. The other, less frequent matrix consisted of loosely arranged collagen fibrils adjacent to the cemental surface. The findings support the notion that, in vitro, a collagenous matrix is formed in contact to diseased and experimentally treated root surfaces. However, the smooth, non-demineralized and scaled cemental surface does not appear to be a suitable substrate for interdigitation with newly produced collagen fibrils.     

De novo organogenesis and plant regeneration in <Emphasis Type="Italic">Pongamia pinnata</Emphasis>, oil producing tree legume

Role of Thidiazuron (TDZ) in inducing adventitious organogenesis in Pongamia was studied. TDZ at different concentrations (0, 0.45, 2.27, 4.54, 6.71, 9.08, 11.35, 13.12 and 22.71 μM) were used for induction of caulogenic bud formation in deembryonated cotyledon explants. Each cotyledon was cut into three segments and identified as proximal, middle and distal. Duration of TDZ exposure, influence of the segment and orientation of the explant were studied. TDZ at 11.35 μM concentration was optimum for the induction of shoots and rapid elongation. Shoots induced at higher concentration elongated after several passages in growth regulator free medium, thereby extending the period of differentiation. Exposure of the explant for 20 days yielded more number of buds than 10 days. Proximal segment of the cotyledon was more responsive. Contact of abaxial surface in the medium was more effective and generated more buds than the adaxial side. Buds differentiated and elongated on transfer to MS basal medium for 8–12 passages of 15 days each. Rooting and elongation of shoots was achieved in charcoal supplemented half-strength MS medium. Rooted plantlets survived on transfer to sand soil mixture. The plants were hardened and transferred to green house. This is the first report on in vitro regeneration of Pongamia pinnata via adventitious organogenesis using TDZ. This protocol may find application in studies in genetic transformation, isolation of somaclonal variants and in induction of mutants. It also provides a system to study the inhibitory role of TDZ on shoot differentiation.     

Three dimensional in vitro culture of preantral follicles following slow-freezing and vitrification of mouse ovarian tissue

To evaluate the effects slow-freezing and vitrification on three dimensional in vitro culture of preantral follicles, ovaries of 12–14 days old female NMRI mice were isolated and randomly assigned to fresh control, slow-freezing and vitrification groups. Slow-freezing was performed using programmable freezer. Vitrification was carried out in a medium consisting of ethylene glycol (EG) and dimethyl sulphoxide (Me₂SO) by needle immersion method. middle sized preantral follicles were mechanically isolated and cultured for 12 days in 0.7% sodium alginate gel. The follicles development and quantitative expression of oocyte specific genes (Bmp15, Gdf9, Fgf8) and the growth related genes (Igf1, Kit, Kit-l) were assessed after 1, 8 and 12 days of culture. Both cryopreserved groups showed reduction of follicular survival rates compared to the control group on days 8 and 12 of culture (P < 0.05). Antrum formation rates reduced in slow-freezing after 12 days of culture (P < 0.05). Evaluation of gene expression showed reduction of Bmp15, Gdf9, Fgf8, Kit and Kit-l during 12 days of culture (P < 0.05). Kit and Kit-l expression in slow-freezing group significantly reduced on day 8 of culture (p < 0.05). Igf1 expression was lower in slow-freezing group on 1st day of culture than vitrification and control groups (P < 0.05). Finally, intergroup comparison showed same expression pattern of genes after 12 days of culture. Thus, cryopreservation of mouse ovaries by both methods can preserve most developmental parameters and expression of maturation genes. However, vitrification is a better method for cryopreservation of mouse ovaries due to greater antrum formation and expression of growth related markers.     

Perfusion culture apparatus for suspended mammalian cells

A variety of processes have been proposed for mammalian cell culture in the commercial production of useful substances (e.g., monoclonal antibodies, therapeutic and diagnostics proteins). Among them, the perfusion culture of suspended non-immobilized cells is the most advantageous. Perfusion culture can be classified by the separation process of suspended cells from the culture mixture into three types, namely filtration, gravitational settling and centrifugation. From a commercial point of view, the present situation and technical problems of suspended-cell perfusion culture will be reviewed based on the three types, The recent development of perfusion culture has been carried out mainly on the filtration separation process, but the centrifugation process seems to have a promising future because of operation stability and scale-up feasibility. The reasons will be explained in details.     

Promotion of indole alkaloid production in Catharanthus roseus cell cultures by rare earth elements

Production of the indole alkaloids, ajmalicine or catharanthine, in cell suspension cultures of Catharanthus roseus was enhanced by cerium (CeO₂ and CeCl₃), yttrium (Y₂O₃) and neodymium (NdCl₃). The yield of ajmalicine in these treated-cultures reached 51 mg l^–1 (CeO₂), 40 mg l^–1 (CeCl₃), 41 mg l^–1 (Y₂O₃) and 49 mg l^–1 (NdCl₃) while catharanthine production reached to 36 mg l^–1 (CeO₂) and 31 mg l^–1 (CeCl₃). A major portion of increased alkaloids was released into medium in these treatments. But Sm₂O₃, SmCl₃, La₂O₃, LaCl₃, complex of chromium (III)-titanium (IV) and NaSeO₄ treatments had little effect on alkaloid production of C. roseus cell cultures.     

Potential pitfalls of the PTV concept in dose-to-medium planning optimization

In typical treatment planning of 3D IMRT, the incident energy fluence is optimized to achieve a homogeneous dose distribution to the PTV. The PTV includes the tumour but also healthy tissues that may have a different dose response for the same incident energy fluence, like bony structures included in the PTV (mandibles in head and neck tumours or femoral bones in sarcomas). Dose to medium optimization compensates for this heterogeneous response, leading to a non-homogeneous energy fluence in the PTV and a non-homogeneous dose in the CTV in the presence of geometric errors. We illustrate qualitatively this statement in a cylindrical geometry where the PTV includes a CTV (7 cm diameter) made of water surrounded by ICRU compact bone (1.2 cm thickness); such configuration was chosen to exaggerate the aforementioned effect. Optimization was performed assuming dose equals photon energy fluence times mass energy absorption coefficient. Bone has a 4% lower dose response in a 6 MV flattening filter free spectrum. After optimization either in medium or assuming everything as water composition, the geometry was shifted by 1.2 cm and dose recomputed. As expected, compensating for the under-response of the bone material during optimization in medium leads to an overdosage of the CTV when patient geometric errors are taken into account. Optimization in dose assuming everything as water composition leads to a uniform coverage. Robust optimization or forcing a uniform atomic composition in the PTV margin may resolve this incompatibility between the PTV concept and dose to medium optimization.