Cocktail Of Factors Research Articles

PROPELLERS OF ORGANISATIONAL SUSTAINABILITY IN HOSPICES IN SOUTH AFRICA

Hospices operating in the non-profit sector are experiencing organisational sustainability pressures, particularly in the wake of the COVID-19 pandemic. Notably, while the population of patients who are terminally ill and requiring palliative care is increasing, the resources required to render this noble service are depleting, so much so that some hospices have closed. The situation raises profound organisational sustainability concerns, and this serves as the motivation for this generic qualitative study to investigate the propellers of organisational sustainability in South African-based hospices operating in the non-profit sector. Data was collected through interviews with twelve senior managers from different hospice organisations. The data was then analysed using Thematic Content Analysis (TCA). Subsequent results reveal that a cocktail of factors, including transparent communication, visionary leadership, a strategic disposition, improved service quality, a strong corporate culture, a diversified revenue base and committed staff, propel organisational sustainability among hospices. Conversely, the COVID-19 pandemicenforced lockdowns and the rampant emergence of many NPOs impede the sustainability prospects of the hospices. With these insights, this study contributes to the organisational sustainability discourse from a uniquely South African perspective, given the dearth of research in this area, particularly in the context of developing economies.

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats.

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 µM insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 µM dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

Abstract WS2-1: Genome editing in human organoids as new models for discovery

Abstract Use of human organoids as new models for discovery. Despite significant advances, there are circumstances where mouse models remain inadequate to model cancer. Human organoid cultures are becoming established as a useful adjunct to mouse models because they enable the propagation of both normal and cancerous tissues in the lab for functional studies, disease modeling, and drug screening. Organoids can be generated from induced pluripotent stem cells or directly from donor tissue samples (e.g. normal breast tissue or tumors). In the latter approach, breast organoid cultures are grown in a hydrogel that mimics the basement membrane and are bathed in a cocktail of growth factors, vitamins, and compounds that mimic the microenvironment provided by stroma, resulting in the growth of cells in a polarized fashion to recapitulate breast acinar- and duct-like structures. Breast epithelial cells within organoids are able to differentiate into all of the major mammary epithelial lineages, and hence organoids can be used to study several types of epithelial lineages and their relative contributions to tumor development. In addition, breast cancer organoids can be used to expand heterogeneous populations of tumor cells for in vitro assays. For example, genome editing approaches can be used to model mutations associated with breast cancer, and compound screening approaches can facilitate target discovery. In this educational talk, we will review some of these recent advances, consider advantages and disadvantages of this model system, and discuss some practical considerations for bio-banking efforts and downstream applications. Citation Format: JM Rosenbluth. Genome editing in human organoids as new models for discovery [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr WS2-1.

Dental Implants in the Geriatric Population: Challenges in Developing Economies

Aging is a natural process that results in biological and physiological changes in the body. geriatric dental patients pose unique challenges that are not routinely seen in younger patients. The difference in bone size, density, shape, and quality present unique opportunities for clinicians to consider when planning for implant replacement therapies in geriatric patients. Despite these setbacks, the popularity of implants in dentistry has risen in recent times and more clinicians and patients see the potentials for oral rehabilitation among the geriatric population. This article attempts to appraise the challenges associated with the provision of dental Implants to the older age group, particularly in developing economies. The challenges of implant treatment in developing countries remain enormous but not insurmountable. As implant treatment for a dental prosthesis in developing countries gains popularity amongst geriatric patients, clinicians must balance this treatment choice on the overall desire and health of the patient, while evaluating his clinical and laboratory competence. Decision on whether or not to place an implant in the mouth of a geriatric patient should not be made on the background of age but on a cocktail of economic, local, and systemic factors.

Peripheral blood-derived monocytes show neuronal properties and integration in immune-deficient rd1 mouse model upon phenotypic differentiation and induction with retinal growth factors

BackgroundCell therapy is one of the most promising therapeutic interventions for retinitis pigmentosa. In the current study, we aimed to assess if peripheral blood-derived monocytes which are highly abundant and accessible could be utilized as a potential candidate for phenotypic differentiation into neuron-like cells.MethodsThe peripheral blood-derived monocytes were reconditioned phenotypically using extrinsic growth factors to induce pluripotency and proliferation. The reconditioned monocytes (RM) were further incubated with a cocktail of growth factors involved in retinal development and growth to induce retinal neuron-like properties. These cells, termed as retinal neuron-like cells (RNLCs) were characterized for their morphological, molecular and functional behaviour in vitro and in vivo.ResultsThe monocytes de-differentiated in vitro and acquired pluripotency with the expression of prominent stem cell markers. Treatment of RM with retinal growth factors led to an upregulation of neuronal and retinal lineage markers and downregulation of myeloid markers. These cells show morphological alterations resembling retinal neuron-like cells and expressed photoreceptor (PR) markers. The induced RNLCs also exhibited relative membrane potential change upon light exposure suggesting that they have gained some neuronal characteristics. Further studies showed that RNLCs could also integrate in an immune-deficient retinitis pigmentosa mouse model NOD.SCID-rd1 upon sub-retinal transplantation. The RNLCs engrafted in the inner nuclear layer (INL) and ganglion cell layer (GCL) of the RP afflicted retina. Mice transplanted with RNLCs showed improvement in depth perception, exploratory behaviour and the optokinetic response.ConclusionsThis proof-of-concept study demonstrates that reconditioned monocytes can be induced to acquire retinal neuron-like properties through differentiation using a defined growth media and can be a potential candidate for cell therapy-based interventions and disease modelling for ocular diseases.

Nicotinamide restricts neural precursor proliferation to enhance catecholaminergic neuronal subtype differentiation from mouse embryonic stem cells.

Emerging evidence indicates that a strong relationship exists between brain regenerative therapies and nutrition. Early life nutrition plays an important role during embryonic brain development, and there are clear consequences to an imbalance in nutritional factors on both the production and survival of mature neuronal populations and the infant’s risk of diseases in later life. Our research and that of others suggest that vitamins play a fundamental role in the formation of neurons and their survival. There is a growing body of evidence that nicotinamide, the water-soluble amide form of vitamin B3, is implicated in the conversion of pluripotent stem cells to clinically relevant cells for regenerative therapies. This study investigated the ability of nicotinamide to promote the development of mature catecholaminergic neuronal populations (associated with Parkinson’s disease) from mouse embryonic stem cells, as well as investigating the underlying mechanisms of nicotinamide’s action. Nicotinamide selectively enhanced the production of tyrosine hydroxylase-expressing neurons and serotonergic neurons from mouse embryonic stem cell cultures (Sox1GFP knock-in 46C cell line). A 5-Ethynyl-2´-deoxyuridine (EdU) assay ascertained that nicotinamide, when added in the initial phase, reduced cell proliferation. Nicotinamide drove tyrosine hydroxylase-expressing neuron differentiation as effectively as an established cocktail of signalling factors, reducing the proliferation of neural progenitors and accelerating neuronal maturation, neurite outgrowth and neurotransmitter expression. These novel findings show that nicotinamide enhanced and enriched catecholaminergic differentiation and inhibited cell proliferation by directing cell cycle arrest in mouse embryonic stem cell cultures, thus driving a critical neural proliferation-to-differentiation switch from neural progenitors to neurons. Further research into the role of vitamin metabolites in embryogenesis will significantly advance cell-based regenerative medicine, and help realize their role as crucial developmental signalling molecules in brain development.

Role of TGF-β/Smad Pathway in the Transcription of Pancreas-Specific Genes During Beta Cell Differentiation.

Autoimmune destruction of pancreatic beta cells causes absolute insulin deficiency and results in type 1 diabetes mellitus (T1DM). The substitution of healthy pancreatic beta cells for damaged cells would be the ideal treatment for T1DM; thus, the generation of pancreatic beta cells from adult stem cells represents an attractive avenue for research. In this study, a cocktail of factors was used to induce the differentiation of pancreatic beta cells from mesenchymal stem cells (MSCs). The differentiation program was divided into five stages, and the roles of the cocktail factors used during each stage were systematically elucidated. Activin A was found to phosphorylate Smad2 and Smad3 in stage III, thereby activating the TGF-β/Smad pathway. Meanwhile, the endocrine-specific transcription factor, Ngn3, and the pancreas-specific miRNAs, miR-375 and miR-26a, were dramatically elevated in stage III. We next demonstrated that Smad4, an important transcription factor in the TGF-β/Smad pathway, could bind to the promoter sequences of target genes and enhance their transcription to initiate the differentiation of beta cells. Use of SB-431542, an inhibitor of the TGF-β/Smad pathway, demonstrated in vivo and in vitro that this pathway plays a critical role in the production of pancreatic beta cells and in modulating insulin secretion. Thus, the TGF-β/Smad pathway is involved in the production of beta cells from adult stem cells by enhancing the transcription of Ngn3, miR-375, and miR-26a. These findings further underline the significant promise of cell transplant therapies for type 1 diabetes mellitus.

Reversible TET Inhibitor As a Hematopoietic Stem Cell Booster: A Novel Strategy to Improve Stem Cell Function

Hematopoietic stem cells (HSCs) are responsible for the adaptation capacity in times of need but are also subjected to disease processes, natural or iatrogenic damage, and age-related attrition. The latter can lead to deficient production, decreased compensatory capacity and degenerative diseases such as MDS. In childhood, hereditary BMF predominates but increasingly with age, acquired idiopathic AA is a leading cause of HSC failure. Throughout life iatrogenic cases and cumulative exposures to environmental toxicities may lead to failure of the HSC compartment. Pharmacologic HSC boosters capable of expanding HSCs would have a wide range of clinical applications in acquired and inherited BMF states, including reconstitution of exhausted hematopoiesis after chemotherapy, aging, or HSC grafting. Currently, hematopoietic growth factors (HGF) are used but lead to progenitor rather than HSC expansion despite their success in clinical application. Depletion of TET2 in murine models leads to impairment of cellular differentiation and increases the proportion of HSCs and progenitors suggesting that TET2 is a key regulator of hematopoietic homeostasis and HSC self-renewal. Alterations of TET2 via somatic mutations and/ or deletion are frequent in MDS whereby HSC and progenitor expansion may be a key component of neoplastic evolution. We hypothesized that chemical agents reversibly inhibiting TET2 activity might phenocopy HSC expansion due to mutations and be applied as HSC boosters. Using structure guided approaches, we designed, synthesized and subsequently optimized bioavailable TET inhibitors (TETi). Among them, the one of the most effective TETi, named TETi76, showed dose-dependent inhibitory activity against TET dioxygenases in in vitro cell-free and cell culture systems with 5hmC production as a read out. We developed esterified forms of TETi76 and used in our cellular experimental models. Esterified TETi is bioavailable and non-toxic to normal bone marrow (BM) cells in therapeutically effective doses. Treatment of TETi76 resulted in a 44+15% and 42+17% increase in the clonogenic potential of human and murine BM cells consistent with the proliferative advantage gained by loss of TET activity in HSCs. We then performed serial replating experiments to determine the effect of TET inhibition on immature hematopoietic progenitor cells. Over 3 consecutive passages, TETi76 treatment prolonged the durability and capacity of human HSCs to maintain colony-forming cells (155+24 vs.107+14 colonies per 1x105 P1 cells). As long-term culture initiating cells (LTC-IC) are the best in vitro surrogates of HSCs, we also investigated the effect of TETi76 in LTC-IC cultures (n=3). The weekly addition of 1μM-TETi76 resulted in nearly 2-fold increase in LTC-IC numbers at the end of the culture (116±27 vs. 64 ±26 colonies per 2x106 P1 cells, p=.011). Expansion of grafts e.g., in the setting of umbilical cord HSC transplant (UCHSC) could be an important medical area of application of TETi76. We performed suspension cultures (n=3) with an optimal cocktail of hematopoietic growth factors (HGF) in the presence or absence of TETi76 (1μM). In control cultures, total cellular output peaked on day 14, but in the presence of TETi, growth continued beyond day 28. Cumulatively, total cellular output per 106 input was 27+2.61 x106cells/mL in control cultures and 32+0.642 x106 cells/mL in TETi treated cultures on day 28. CD34+ cell output was significantly higher in cultures treated with TETi vs. vehicle (2.5x105vs. 0.9X106)CD34+ cells per 1x104 CD34+ cell input). Similar effects were observed in murine BM suspension cultures. BM cells from C57BL/6 mice (n=3) were supplemented with HGF± TETi76 (1μM). TETi treatment led to a 5-fold HSC expansion compared to vehicle treated cells at 20 days of culture. The effect of TETi76 was reversed by treatment with ascorbic acid (50 μM), a known TET activator. Cumulatively our in vitro results suggest that the presence of TETi prevents exhaustion of immature cells, observed with growth factor driven expansion. In summary, our study indicates that novel agents modulating TET activity prevent exhaustion of HSC and may help expand the HSC in vitro. In vivo experiments examining the effects of TETi on hematopoietic recovery following radiation-induced aplasia, and competitive transplant experiments of grafts exposed in vivo and in vitro to TETi are underway. Disclosures Sekeres: Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.

Stem Cells in theMammalian Gonads.

Besides spermatogonial stem cells (SSCs) and ovarian stem cells (OSCs), a novel population of pluripotent stem cells termed very small embryonic-like stem cells (VSELs) has been reported in both adult mouse and human testes and ovaries. VSELs and SSCs/OSCs are developmentally linked to each other. VSELs are relatively quiescent, small-sized stem cells that undergo asymmetrical cell divisions (ACDs) whereby they self-renew and give rise to the slightly bigger SSCs/OSCs which in turn undergo symmetrical cell divisions (SCDs) and clonal expansion to form germ cell chains/nests before further differentiation into gametes. Comparison of VSELs and SSCs/OSCs for their potential to differentiate into sperm/oocytes is irrelevant since VSELs only undergo ACD to give rise to SSCs/OSCs that further differentiate into gametes. Being relatively quiescent, VSELs survive oncotherapy and can be manipulated to regenerate nonfunctional gonads of cancer survivors, and thus there is possibly no need to bank testicular/ovarian tissue prior to oncotherapy. Being developmentally linked to the primordial germ cells (PGCs) which are the natural precursors to the gametes, VSELs differentiate into haploid sperm/oocyte-like structures in vitro when cultured on appropriate feeder support, in the absence of a cocktail of growth factors. VSELs express receptors for pituitary and sex hormones (FSHR, ER) and thus get directly stimulated/affected by their circulating levels. Excessive self-renewal of VSELs in the gonads may initiate testicular and ovarian cancers. To conclude, VSELs can be targeted to regenerate the gonads of patients with gonadal insufficiency including cancer survivors and are excellent candidates to differentiate into gametes in vitro.

Functional Differentiation of Neural Stem Cells into Neuronal Subtypes: A Biological Tool for Developmental Neurotoxicity Studies

Neural Stem Cells (NSCs), owing to their potential to get differentiated into various mature cell subtypes including neuronal cells have proved to be an indefinite source of 'raw material' for their application in developmental neurotoxicity as well as therapeutic intervention in neurodegenerative disorders. However, applications of NSCs for such purposes have been broadly limited by lack of enough methods for their directed differentiation. Herein, we describe a chemically defined protocol for efficient differentiation of rat neural stem cells to neuronal subtypes using an 8-day time period. NSCs, subject to NGF (50 ng/mL) were differentiated into neuronal sub-types supplemented with a cocktail of growth factors and supplements. Differentiating cells revealed a gradual and significant induction in the neuronal markers and a parallel decrease in markers of stemness as confirmed by immunocytochemical and translational analysis. The expression of markers was found to be maximum at day 8 of differentiation. Such selective differentiation of NSCs into neurons could offer an imperative step towards generation of NSC derivatives that could facilitate their utilization for research studies.

185 Directed Differentiation of Porcine Induced Pluripotent Stem Cells into all Three Germ Layers via Embryoid Body Formation

Human induced pluripotent stem cells (iPSC) have been generated by reprogramming somatic cells using a cocktail of stem cell transcription factors but the application has been limited in transplantation therapies. The pig represents an ideal model for human clinical research, in part because of its similarity to human physiology and immunology but also because of its use in assessing side effects in long-term preclinical studies. Porcine induced pluripotent stem cells (piPSC) have been established in many studies but their differentiation pattern has not been reported. The aim of this study was to estimate the efficiency and pattern of differentiated piPSC into all 3 germ layers using embryoid body (EB) formation. Two piPSC lines (VSMUi001-A and VSMUi001-D) were induced from porcine embryonic fibroblasts by retroviral overexpression of 5 human reprogramming transcription factors (OCT4, SOX2, KLF4, c-MYC, and LIN28). For EB formation, the piPSC were harvested by treating with TrypLE™ Select (Thermo Fisher Scientific, Waltham, MA, USA) and the cells were cultured in nonadherent 96-well plates in piPSC media without growth factors. Data are expressed as mean ± SEM of at least 3 independent experiments. Statistical analyses were evaluated with Student t-tests for comparison between the 2 cell lines. Statistical significance was set at a P-value of < 0.05. The percentages of EB formation, which were calculated as the number of wells containing EB on Day 3 of differentiation, were 95.3 ± 3.42 and 89.1 ± 5.34 (VSMUi001-A and VSMUi001-D, respectively). However, there was no significant difference between the percentages of EB formation derived from the 2 cell lines. For EB size measurement, 20 EB per experiment were taken after incubation for 3, 7, 14, and 21 days. Both EB sizes increased over time (average diameter of 238.1 ± 6.18, 297.9 ± 4.10, 438.6 ± 13.33, and 728.8 ± 24.92 mm from VSMUi001-A, and 255.8 ± 5.12, 357.9 ± 3.94, 459.6 ± 11.88, and 439.4 ± 20.31 mm from VSMUi001-D). Moreover, both EB displayed homogeneity in size and shape (Day 3, 7), exhibited a cystic structure (Day 14), and a vesicular cavity was present (Day 21). For immunohistochemical analysis, both EB had lower levels of cleaved caspase 3, a marker of apoptotic cells, on Day 3 but higher levels of cleaved caspase 3 from Day 7 through 21. On the contrary, EB showed higher levels of Ki67, a marker of proliferating cells, on Day 3 but lower levels of Ki67 on Days 7, 14, and 21, respectively. In gene expression assessment, EB exhibited ectoderm gene (NeuroD1), mesoderm genes (TNNT2 and TNNI1), and endoderm genes (SOX17 and Endolase) at Day 7 and 21 by using RT-PCR. In conclusion, we report the successful in vitro formation of cystic EB from 2 piPSC lines, indicating that the piPSC could differentiate into 3 germ layers. This will allow researchers to unveil the roadmap of molecular cues needed for piPSC differentiation. This research project is supported by grants from the Mahidol University, Thailand.

Protein Kinase-A Inhibition Is Sufficient to Support Human Neural Stem Cells Self-Renewal.

Human pluripotent stem cell-derived neural stem cells offer unprecedented opportunities for producing specific types of neurons for several biomedical applications. However, to achieve it, protocols of production and amplification of human neural stem cells need to be standardized, cost effective, and safe. This means that small molecules should progressively replace the use of media containing cocktails of protein-based growth factors. Here we have conducted a phenotypical screening to identify pathways involved in the regulation of hNSC self-renewal. We analyzed 80 small molecules acting as kinase inhibitors and identified compounds of the 5-isoquinolinesulfonamide family, described as protein kinase A (PKA) and protein kinase G inhibitors, as candidates to support hNSC self-renewal. Investigating the mode of action of these compounds, we found that modulation of PKA activity was central in controlling the choice between self-renewal or terminal neuronal differentiation of hNSC. We finally demonstrated that the pharmacological inhibition of PKA using the small molecule HA1004 was sufficient to support the full derivation, propagation, and long-term maintenance of stable hNSC in absence of any other extrinsic signals. Our results indicated that tuning of PKA activity is a core mechanism regulating hNSC self-renewal and differentiation and delineate the minimal culture media requirement to maintain undifferentiated hNSC in vitro.

Poststroke angiogenesis: blood, bloom, or brood?

Angiogenesis, the formation of new capillaries from existing blood vessels, is a naturally occurring phenomenon in adult humans, encompassing a range of normal functions from the physiological maturation of the female reproductive system to wound healing, as well as pathological functions, such as aiding unwanted tumor growth. Although ample evidence suggests that angiogenesis occurs in humans and laboratory animals after brain ischemia,1,2 it has long been debated whether angiogenesis contributes to the recovery of function after stroke and whether it can be harnessed as a therapy to treat stroke. The pro and con opinions of poststroke angiogenesis have been eloquently and pertinently discussed by Dr Greenberg, who attests to the benefit of angiogenesis and the potential for clinical application and by Drs Adamczak and Hoehn, who address the risk of angiogenesis and potential consequences in aggravating stroke progression. In light of the recent …

Role of Erythropoietin and Other Growth Factors inEx VivoErythropoiesis

Erythropoiesis is a vital process governed through various factors. There is extreme unavailability of suitable donor due to rare phenotypic blood groups and other related complications like hemoglobinopathies, polytransfusion patients, and polyimmunization. Looking at the worldwide scarcity of blood, especially in low income countries and the battlefield, mimicking erythropoiesis usingex vivomethods can provide an efficient answer to various problems associated with present donor derived blood supply system. Fortunately, there are manyex vivoerythropoiesis methodologies being developed by various research groups using stem cells as the major source material for large scale blood production. Most of theseex vivoprotocols use a cocktail of similar growth factors under overlapping growth conditions. Erythropoietin (EPO) is a key regulator in mostex vivoprotocols along with other growth factors such as SCF, IL-3, IGF-1, and Flt-3. Now transfusable units of blood can be produced by using these protocols with their set of own limitations. The present paper focuses on the molecular mechanism and significance of various growth factors in these protocols that shall remain helpful for large scale production.

Into the groove

Adult cells can be routinely reprogrammed into pluripotent stem cells by chemical and genetic means, such as the expression of a cocktail of exogenous transcription factors. It is now shown that growing cells on substrates with aligned features such as microgrooves can enhance this process.

Identification Of CD34+ Microparticles In Cord Blood Units

Introduction Cell-derived microparticles (MPs), small membrane vesicles, are considered as markers of cell activation, as well as apoptosis and recently was shown to participate in important normal biological functions and pathological conditions associated mainly with endothelial injury, thrombosis and inflammation. Recently platelet MPs have been detected in patients undergoing allogeneic stem cell transplantation. Umbilical cord blood is an alternative stem cell source for transplantation where the presence of MPs has not been extensively studied. The aim of this study is to quantify the MPs of CD34+ cells in cord blood units and to investigate their relationship with different parameters. Methods Cord blood units (CBUs) (n=41) were processed using the Sepax automated method (Biosafe). After processing, cryopreservation was performed using 10% DMSO in a controled rate freezer (IceCube, Sy-lab). Units were thawed after two weeks. Adjusted variables of the CBUs included their net volume, the absolute number of CD34+ per graft and their viability as determined by flow cytometry, the number of leukocytes as recorded with an hematology analyzer and the absolute number of erythroblasts as estimated by optical microscope after Giemsa staining. Mononuclear cells were seeded in semisolid cultures in the presence of a cocktail of growth factors for colony forming unit granulocyte/macrophage (CFU-GM), burst forming unit erythroid (BFU-E) and Colony-Forming Unit-Granulocyte, Erythrocyte, Monocyte/macrophage, Megakaryocyte (CFU-GEMM) colony growth. The MPs were isolated after centrifugation of the plasma and their number was determined after incubation with Annexin V and CD34 by flow cytometry. Electron microscope photographs were obtained, after the proceedings for TEM. Statistical analysis was done using t-test, Pearson's and Spearman's depending on the normality of the distribution of variables. Results The mean number of AnnV+/CD34+MPs increased during post-processing (76,7±15,9) compared to the number of pre-processing AnnV+/CD34+MPs (73±22 ) and decreased during post-thawing (52,4 ±16,7). On univariate analysis found that the number of AnnV+/CD34+MPs (pre-processing vs. post-processing) has statistically significant mean positive correlation (p<0,012, Pearson's rho =0,823) whereas the number of MPs post-thawing was not statistically significant. Pre-processing AnnV+/CD34+MPs has statistically significant correlation with the absolute number of pre-processing CD34+ cell per unit (p<0,0001, Spearman's rho =0,835), post-processing (p<0,0001, Spearman's rho =0,814) and post-thawing CD34+ cells per unit (p<0,0001, Spearman's rho =0,993). Correlation of post-processing CFUs and AnnV+/CD34+MPs showed that the number of AnnV+/CD34+MPs has statistically significant mean negative correlation to the number of CFU-GEMM (p<0,028, Spearman's rho = -0,348). Conclusions These data confirm the identification of stem cell-derived MPs from cord blood samples. Moreover, the number of pre- and post- processing AnnV+/CD34+MPs was statistically correlated. In addition, the absolute number of CD34 (pre-, post- processing and post-thawing) was associated with the AnnV+/CD34+MPs pre- processing. Thus it remains to be shown if the stem cell derived MPs could be a useful parameter to predict the quality of the cord blood unit. Disclosures: No relevant conflicts of interest to declare.

Directing Smooth Muscle Cell Fate

Cellular reprogramming has opened new doors for scientists to study human disease and develop methods for diagnosis and therapy. Cellular reprogramming has been around since first achieving somatic nuclear reprogramming in 1962, and identifying a single transcription factor could convert fibroblasts into muscle in 1986.1,2 But in 2006, the reprogramming field was suddenly transformed when Shinya Yamanaka discovered that ectopic expression of only 4 transcription factors consisting of OCT4, SOX2, KLF4, and c-MYC (OSKM) could transform skin fibroblasts into an embryonic stem cell–like fate, aka, induced pluripotent stem (iPS) cells that have the potential to become any cell type in the body.3 The realization that a cell’s fate is reversible through defined and simple genetic manipulation created a wave of new direct reprogramming studies—reprogramming fibroblasts into an alternative mature cell type without passing through a pluripotent state—to search for genetic cocktails to manipulate and direct cell fate. Studies performed by multiple groups elegantly showed that a cocktail of 3 cardiac transcription factors important during cardiac development could directly reprogram cardiac fibroblasts into cardiomyocyte-like cells in vitro.4–7 Furthermore, the reprogramming process was more effective in endogenous cardiac fibroblasts after injury in a mouse model.6,8 As direct reprogramming of fibroblasts into cardiomyocytes and neurons has been successful,4–9 it is thought that other mature cell types can be generated from fibroblasts if given the right cocktail of factors. Article, see p 1433 In the meantime, while researchers search for the right cocktail to drive direct differentiation for each cell fate, alternative cell reprogramming approaches have emerged, which provide a faster, safer, and more efficient strategy than the current iPS technology. These approaches involve shortcuts to reprogram fibroblasts through a process of partial reprogramming. Rather than …

Stem cell bioengineering strategies to widen the therapeutic applications of haematopoietic stem/progenitor cells from umbilical cord blood

Umbilical cord blood (UCB) transplantation has observed a significant increase in recent years, due to the unique features of UCB haematopoietic stem/progenitor cells (HSCs) for the treatment of blood-related disorders. However, the low cell numbers available per UCB unit significantly impairs the widespread use of this source for transplantation of adult patients, resulting in graft failure, delayed engraftment and delayed immune reconstitution. In order to overcome this issue, distinct approaches are now being considered in clinical trials, such as double-UCB transplantation, intrabone injection or ex vivo expansion. In this article the authors review the current state of the art, future trends and challenges on the ex vivo expansion of UCB HSCs, focusing on culture parameters affecting the yield and quality of the expanded HSC grafts: novel HSC selection schemes prior to cell culture, cytokine/growth factor cocktails, the impact of biochemical factors (e.g. O2 ) or the addition of supportive cells, e.g. mesenchymal stem/stromal cell (MSC)-based feeder layers) were addressed. Importantly, a critical challenge in cellular therapy is still the scalability, reproducibility and control of the expansion process, in order to meet the clinical requirements for therapeutic applications. Efficient design of bioreactor systems and operation modes are now the focus of many bioengineers, integrating the increasing 'know-how' on HSC biology and physiology, while complying with the GMP standards for the production of cellular products, i.e. through the use of commercially available, highly controlled, disposable technologies.

Human amniotic fluid stem cells have a potential to recover ovarian function in mice with chemotherapy-induced sterility

BackgroundHuman amniotic fluid cells (hAFCs) may differentiate into multiple cell lineages and thus have a great potential to become a donor cell source for regenerative medicine. The ability of hAFCs to differentiate into germ cell and oocyte-like cells has been previously documented. Herein we report the potential use of hAFCs to help restore follicles in clinical condition involving premature ovarian failure.ResultsHuman amniotic fluid was obtained via amniocentesis, yielding a subpopulation of cloned hAFCs that was able to form embryoid bodies (EBs) and differentiate into three embryonic germ layers. Moreover, culture of EBs in medium containing human follicular fluid (HFF) or a germ cell maturation factor cocktail (FAC), expressed germ cells markers such as BLIMP1, STELLA, DAZL, VASA, STRA8, SCP3, SCP1, and GDF9. Furthermore, one cell line was grown from clone cells transfected with lentivirus-GFP and displaying morphological characteristics of mesenchymal cells, had the ability to restore ovarian morphology following cell injection into the ovaries of mice sterilized by intraperitoneal injection of cyclophosphamide and busulphan. Restored ovaries displayed many follicle-enclosed oocytes at all stages of development, but no oocytes or follicles were observed in sterilized mice whose ovaries had been injected with medium only (control). Notably, identification of GFP-labeled cells and immunostaining with anti–human antigen-specific antibodies demonstrated that grafted hAFCs survived and differentiated into granulosa cells which directed oocyte maturation. Furthermore, labeling of ovarian tissue for anti-Müllerian hormone expression, a functional marker of folliculogenesis, was strong in hAFCs-transplanted ovaries but inexistent in negative controls.ConclusionThese findings highlight the possibility of using human amniotic fluid-derived stem cells in regenerative medicine, in particular in the area of reproductive health.

Expansion and differentiation of human primary osteoblasts in two- and three-dimensional culture

Despite the regenerative capability of bone, treatment of large defects often requires bone grafts. The challenge for bone grafting is to establish rapid and sufficient vascularization. Three-dimensional (3D) multicellular spheroids consisting of the relevant cell types can be used as “mini tissues” to study the complexity of angiogenesis. We investigated two-dimensional (2D) expansion, differentiation and characterization of primary osteoblasts as steps toward the establishment of 3D multicellular spheroids. Supplementation of cell culture medium with vitamin D3 induces the osteocalcin expression of osteoblasts. An increased osteocalcin concentration of 10.8 ± 0.58 ng/ml could be measured after 19 days in supplemented medium. Vitamin D3 has no influence on the expression of alkaline phosphatase or the deposition of calcium. Expression of these additional osteogenic markers requires addition of a cocktail of osteogenic factors that, conversely, have no influence on the expression of osteocalcin. Supplementation of the cell culture medium with both vitamin D3 and a cocktail of osteogenic factors is recommended to produce an osteoblast phenotype that secretes osteocalcin, expresses alkaline phosphatase and deposits calcium. In such a supplemented medium, a mean osteocalcin concentration of 11.63 ± 4.85 ng/ml was secreted by the osteoblasts. Distinguishing osteoblasts and fibroblasts remains a challenge. Neither differentiated nor undifferentiated osteoblasts can be distinguished from fibroblasts by the expression of CD90, ED-A-fibronectin or α-smooth muscle actin; however, these cell types exhibit clear differences in their growth characteristics. Osteoblasts can be arranged as 3D spheroids by coating the bottom of the cell culture device with agarose. The cellular composition of 3D multicellular spheroids can be evaluated quantitatively using vital fluorescence labeling techniques. Spheroids are a promising tool for studying angiogenic and osteogenic phenomena in vivo and in vitro.