Peak MSC-Are We There Yet?

Human mesenchymal stem cells (hMSCs) are a critical raw material for many regenerative medicine products, including cell-based therapies, engineered tissues, or combination products, and are on the brink of radically changing how the world of medicine operates. Their unique characteristics, potential to treat many indications, and established safety profile in more than 800 clinical trials have contributed to their current consumption and will only fuel future demand. Given the large target patient populations with typical dose sizes of 10's to 100's of millions of cells per patient, and engineered tissues being constructed with 100's of millions to billions of cells, an unprecedented demand has been created for hMSCs. The fulfillment of this demand faces an uphill challenge in the limited availability of large quantities of pharmaceutical grade hMSCs for the industry-fueling the need for parallel rapid advancements in the biomanufacturing of this living critical raw material. Simply put, hMSCs are no different than technologies like transistors, as they are a highly technical and modular product that requires stringent control over manufacturing that can allow for high quality and consistent performance. As hMSC manufacturing processes are optimized, it predicts a future time of abundance for hMSCs, where scientists and researchers around the world will have access to a consistent and readily available supply of high quality, standardized, and economical pharmaceutical grade product to buy off the shelf for their applications and drive product development-this is "Peak MSC."

Pseudoislets in stirred-suspension culture exhibit enhanced cell survival, propagation and insulin secretion.

Cells from primary islets and beta-cell lines form pseudoislets (PIs) in static cultures. Interestingly, MIN6 beta-cells with aberrant regulation of proliferation form PIs which cease to grow after a week in culture. This growth arrest is attributed to a pro-apoptotic and anti-proliferative PI environment. We hypothesized that cell necrosis due to poor nutrient transport in dishes rather than apoptosis effects the observed PI size restriction. Formation of beta-cell PIs was explored in stirred-suspension bioreactors with enhanced mass transfer. Cells in stirred-suspension proliferated continuously and the PI size increased for two weeks. Bioreactor PIs displayed regulated basal insulin secretion and enhanced responsivity to glucose and incretins. Compared to dishes, cell viability in the bioreactor was higher with lower released lactate dehydrogenase activity. Similar expression of p21 and p27 in monolayers and PIs did not suggest an anti-proliferative PI milieu. Caspase-2, -8 and -9 activities were comparable in dish and bioreactor PIs, and the latter continued to grow after one week of culture. Thus, apoptosis is not sufficient to explain the differences in PI size between dishes and bioreactor. Moreover, the bioreactor method described here may be used to generate PIs with increased cell viability and function for research and clinical applications.

Scalable stirred-suspension bioreactor culture of human pluripotent stem cells.

Advances in stem cell biology have afforded promising results for the generation of various cell types for therapies against devastating diseases. However, a prerequisite for realizing the therapeutic potential of stem cells is the development of bioprocesses for the production of stem cell progeny in quantities that satisfy clinical demands. Recent reports on the expansion and directed differentiation of human embryonic stem cells (hESCs) in scalable stirred-suspension bioreactors (SSBs) demonstrated that large-scale production of therapeutically useful hESC progeny is feasible with current state-of-the-art culture technologies. Stem cells have been cultured in SSBs as aggregates, in microcarrier suspension and after encapsulation. The various modes in which SSBs can be employed for the cultivation of hESCs and human induced pluripotent stem cells (hiPSCs) are described. To that end, this is the first account of hiPSC cultivation in a microcarrier stirred-suspension system. Given that cultured stem cells and their differentiated progeny are the actual products used in tissue engineering and cell therapies, the impact of bioreactor's operating conditions on stem cell self-renewal and commitment should be considered. The effects of variables specific to SSB operation on stem cell physiology are discussed. Finally, major challenges are presented which remain to be addressed before the mainstream use of SSBs for the large-scale culture of hESCs and hiPSCs.

Expansion and differentiation of human embryonic stem cells to endoderm progeny in a microcarrier stirred-suspension culture.

Embryonic stem cells (ESCs) with their abilities for extensive proliferation and multi-lineage differentiation can serve as a renewable source of cellular material in regenerative medicine. However, the development of processes for large-scale generation of human ESCs (hESCs) or their progeny will be necessary before hESC-based therapies become a reality. We hypothesized that microcarrier stirred-suspension bioreactors characterized by scalability, straightforward operation, and tight control of the culture environment can be used for hESC culture and directed differentiation. Under appropriate conditions, the concentration of hESCs cultured in a microcarrier bioreactor increased 34- to 45-fold over 8 days. The cells retained the expression of pluripotency markers such as OCT3/4A, NANOG, and SSEA4, as assessed by quantitative PCR, immunocytochemistry, and flow cytometry. We further hypothesized that hESCs on microcarriers can be induced to definitive endoderm (DE) when incubated with physiologically relevant factors. In contrast to embryoid body cultures, all hESCs on microcarriers are exposed to soluble stimuli in the bulk medium facilitating efficient transition to DE. After reaching a peak concentration, hESCs in microcarrier cultures were incubated in medium containing activin A, Wnt3a, and low concentration of serum. More than 80% of differentiated hESCs coexpressed FOXA2 and SOX17 in addition to other DE markers, whereas the expression of non-DE genes was either absent or minimal. We also demonstrate that the hESC-to-DE induction in microcarrier cultures is scalable. Our findings support the use of microcarrier bioreactors for the generation of endoderm progeny from hESCs including pancreatic islets and liver cells in therapeutically useful quantities.

Propagation of embryonic stem cells in stirred suspension without serum.

Embryonic stem cells (ESCs) with their unlimited capacity for self-renewal and ability to differentiate along multiple cell lineages are a superb starting material for biotechnology applications and cellular therapies. However, realization of the potential of ESCs requires the development of scalable systems for their production in large quantities and in a regulated manner. Here, we describe a methodology for the expansion of mouse ESCs (mESCs) as pluripotent aggregates in a stirred suspension bioreactor and in medium without serum. Initially, the culture of feeder cell-independent mESCs in dishes was adapted to serum-free conditions. Also, we explored whether spinner flasks equipped with a triangle-shaped impeller and baffles support the culture of mESC aggregates. Serum-free culture in these vessels resulted in an almost 20-fold increase in the live mESC concentration over 4 days without significant loss of cell viability. Even after consecutive passages, mESCs retained high expression of pluripotency markers Oct3/4, Rex1 and SSEA-1. More importantly, when differentiation was induced these cells adopted fates of all three germ layers namely neuroectoderm, cardiac mesoderm and definitive endoderm. These findings demonstrate that stem cells can be propagated under serum-free conditions in a scalable stirred-suspension culture without loss of their pluripotency.

Standardized biosynthesis of flavan-3-ols with effects on pancreatic beta-cell insulin secretion.

Flavan-3-ols, such as green tea catechins represent a major group of phenolic compounds with significant medicinal properties. We describe the construction and optimization of Escherichia coli recombinant strains for the production of mono- and dihydroxylated catechins from their flavanone and phenylpropanoid acid precursors. Use of glucose minimal medium, Fe(II), and control of oxygen availability during shake-flask experiments resulted in production yield increases. Additional production improvement resulted from the use of medium rather than high-copy number plasmids and, in the case of mono-hydroxylated compounds, the addition of extracellular cofactors in the culture medium. The established metabolic engineering approach allowed the biosynthesis of natural catechins at high purity for assessing their possible insulinotropic effects in pancreatic beta-cell cultures. We demonstrated that (+)-afzelechin and (+)-catechin modulated the secretion of insulin by pancreatic beta-cells. These results indicate the potential of applying metabolic engineering approaches for the synthesis of natural and non-natural catechin analogues as drug candidates in diabetes treatments.

Stem/Progenitor cell sources of insulin-producing cells for the treatment of diabetes.

Patients with diabetes experience decreased insulin secretion that is linked to a significant reduction in the number of islet cells. Reversal of diabetes can be achieved through islet transplantation, but the scarcity of donor islets severely hinders wide application of this therapeutic modality. Toward that end, embryonic stem cells, adult tissue-residing progenitor cells, and regenerating native beta-cells may serve as sources of islet cell surrogates. Insulin-producing cells generated from stem or progenitor cells display subsets of native beta-cell attributes, indicating the need for further development of methods for differentiation to completely functional beta-cells. Pharmacological approaches aiming at stimulating the in vivo/ex vivo regeneration of beta-cells have also been proposed as a way of augmenting islet cell mass. We review the current state of the generation of insulin-producing cells from different sources with emphasis on embryonic stem cells and adult progenitor cells. Challenges for the clinical use of these sources are also discussed.

Predicting memory performance in normal ageing using different measures of hippocampal size.

A number of different methods have been employed to correct hippocampal volumes for individual variation in head size. Researchers have previously used qualitative visual inspection to gauge hippocampal atrophy. The purpose of this study was to determine the best measure(s) of hippocampal size for predicting memory functioning in 102 community-dwelling individuals over 80 years of age. Hippocampal size was estimated using magnetic resonance imaging (MRI) volumetry and qualitative visual assessment. Right and left hippocampal volumes were adjusted by three different estimates of head size: total intracranial volume (TICV), whole-brain volume including ventricles (WB+V) and a more refined measure of whole-brain volume with ventricles extracted (WB). We compared the relative efficacy of these three volumetric adjustment methods and visual ratings of hippocampal size in predicting memory performance using linear regression. All four measures of hippocampal size were significant predictors of memory performance. TICV-adjusted volumes performed most poorly in accounting for variance in memory scores. Hippocampal volumes adjusted by either measure of whole-brain volume performed equally well, although qualitative visual ratings of the hippocampus were at least as effective as the volumetric measures in predicting memory performance in community-dwelling individuals in the ninth or tenth decade of life.

Comparing white matter lesions on T2 and FLAIR MRI in the Sydney Older Persons Study.

There is suggestion that magnetic resonance imaging (MRI) fluid-attenuated inversion recovery (FLAIR) sequence may be more accurate than T2 images in detecting white matter lesions (WML) in older people. Comparative ratings of these two image sequences have not been directly investigated in very old individuals to date. We compared the ratings of periventricular and deep WML on these two sequences in a sample of 111 community dwellers (mean age 85.5 years) using semiquantitative methods. Periventricular WML were as commonly detected on T2 as on FLAIR but were more severely rated on the latter sequence. No such bias was observed for the deep WML. With one exception, correlations between the two sets of measures were significant at the P < 0.001 level (range: 0.34-0.75). Intrarater reliability coefficients were moderate to excellent for most ratings. These results suggest that ratings performed on T2-weighted images to detect WML in very old individuals are very comparable with those performed on FLAIR images although FLAIR may allow a finer grading of periventricular lesions. Absence of FLAIR does not preclude the identification of WML in this population. These findings have clinical and epidemiological relevance where the acquisition of supplementary MRI data may not always be possible.

Hippocampal size and memory function in the ninth and tenth decades of life: the Sydney Older Persons Study.

OBJECTIVES: The purpose of this study was to define magnetic resonance imaging (MRI) correlates of normal brain ageing, with the specific objective of investigating whether the size of the hippocampus is selectively correlated with age related memory performance in non-demented individuals in their ninth and tenth decades of life. METHODS: Hippocampal size was estimated using MRI based volumetry and qualitative visual assessment in 102 community dwelling individuals aged between 81 and 94 years. Participants were evaluated on a variety of clinical and experimental instruments, including a comprehensive neuropsychological test battery. All participants underwent neurological examination, an extensive medical history was obtained, and an informant confirmed details of each participant's functional ability. RESULTS: Both visual and volumetric hippocampal measures were identified as robust predictors of memory performance, even when the influence of age related illnesses and sociodemographic variables was accounted for. When the sample was reduced to include the most cognitively healthy participants who were rated by an informant as showing no evidence of cognitive decline, the left hippocampal measures remained significant predictors of delayed retention of verbal material. CONCLUSIONS: These findings suggest that hippocampal volumes are selectively correlated with memory functioning in both normal and successful ageing.

Hemi-orolingual angioedema and ACE inhibition after alteplase treatment of stroke.

One hundred seventy-six consecutive patients treated with IV tissue plasminogen activator (tPA) for acute ischemic stroke were examined prospectively, and orolingual angioedema was found in nine (5.1%; 95% CI 2.3 to 9.5). The reaction was typically mild, transient, and contralateral to the ischemic hemisphere. Risk of angioedema was associated with angiotensin-converting enzyme inhibitors (relative risk [RR] 13.6; 95% CI 3.0 to 62.7) and signs on initial CT of ischemia in the insular and frontal cortex (RR 9.1; 95% CI 1.4 to 30.0).

Neuropsychological manifestations in a case of bilateral thalamic infarction.

Neuropsychological manifestation has been reported with lesions of the anterior and non-specific thalamic nuclei and mammilothalamic tract (MMT). These have been reported in the setting of arterial infarction and/or haemorrhage. Cerebral venous sinus thrombosis (CVT) is a rare cause of brain infarction. It occurs in the setting of oral contraceptive administration or pregnancy. Inherited thrombophilias are documented risk factors. The most frequent being heterozygous factor V Leiden mutation. We report a single case of bilateral thalamic infarction due to cerebral vein and sinus thrombosis. Clinically the case manifested with memory impairment and dysexecutive symptoms. Predisposing factor for venous thrombosis was a homozygous factor V Leiden mutation. The patient was treated with anticoagulation and made a good recovery.

Traumatic brain injury as a risk factor for Alzheimer's disease: a review.

Accumulating epidemiological evidence implicates traumatic brain injury as a pathogenic agent in the development of Alzheimer's disease (AD). Considering the increase in the prevalence of both traumatic brain injury and AD in recent times, the possibility that brain trauma may provoke the early development of AD has important implications for health service planning, preventative efforts, and medico-legal compensation settlements. This paper evaluates the plausibility of the proposed link between traumatic brain injury and AD, largely by way of exploring a theoretical perspective advanced by Satz (1993) and considering recent contributions from the epidemiological, neuropathological, and biochemical literature that are pertinent to this issue. The literature reviewed provides sufficient support and empirical vindication to give credence to the proposed association between these two neuropsychological entities at the statistical, theoretical, and biological level.

The significance of the fovea palantini in complete denture prosthodontics.

Clinical, radiographic, and histologic studies of the fovea palatini indicate that they were positioned 1.31 mm. (mean of 100 subjects) in front of the vibrating line. Radiographically and histologically, the foveae were located in soft tissue covering the hard palate in all specimens. Histologically, complex nerve endings were found just anterior to the foveae and spreading to the soft palate. The findings were related to clinical aspects of complete dentures.