Representations of lipid nanoparticles using large language models for transfection efficiency prediction.

MOTIVATION: Lipid nanoparticles (LNPs) are the most widely used vehicles for mRNA vaccine delivery. The structure of the lipids composing the LNPs can have a major impact on the effectiveness of the mRNA payload. Several properties should be optimized to improve delivery and expression including biodegradability, synthetic accessibility and transfection efficiency (TE). RESULTS: To optimize LNPs we developed and tested models that enable the virtual screening of LNPs with high TE. Our best method uses the lipid SMILES as inputs to a Large Language Model (LLM). LLM generated embeddings are then used by a downstream gradient-boosting classifier. As we show, our method can more accurately predict lipid properties which could lead to higher efficiency and reduced experimental time and costs. AVAILABILITY AND IMPLEMENTATION: Code and data links available at: https://github.com/Sanofi-Public/LipoBART.

A detailed analysis of possible efficacy signals of NTHi-Mcat vaccine against severe COPD exacerbations in a previously reported randomised phase 2b trial.

BACKGROUND: An investigational vaccine containing non-typeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis (Mcat) surface proteins did not show vaccine efficacy (VE) against combined moderate and severe (moderate/severe) exacerbations in a randomised, observer-blinded, placebo-controlled phase 2b trial of patients with chronic obstructive pulmonary disease (COPD). Nevertheless, observations on rates of severe exacerbations and hospitalisations encouraged further evaluation. METHODS: Patients with stable COPD (moderate to very severe airflow limitation, Global Initiative for Chronic Obstructive Lung Disease [GOLD] stage 2-4), 40-80 years and at least one moderate/severe exacerbation in the last year received two doses of NTHi-Mcat vaccine or placebo plus standard care. Secondary analyses were conducted on VE against exacerbations according to severity. Potential predictive factors at baseline for VE against severe exacerbations were explored in post-hoc analyses. RESULTS: Of 606 patients enrolled, 571 were included in the efficacy analysis (279 in NTHi-Mcat vaccine group, 292 in placebo group). VE against severe acute exacerbations of COPD (AECOPD) in various subgroups was 52.11 % (p = 0.015; frequent exacerbators), 65.43 % (p = 0.015; baseline GOLD grade 4), 38.24 % (p = 0.034; previous pneumococcal and/or influenza vaccination). VE was 52.49 % (p = 0.044) for the 6-12 months period after 1 month post-dose 2. Multivariable analysis identified two factors (frequent exacerbator status plus inhaled corticosteroid use at baseline) associated with significant VE against severe AECOPD; in this subpopulation, VE was 74.99 % (p < 0.001). CONCLUSION: Results suggest potential efficacy with the NTHi-Mcat vaccine against severe exacerbations in certain patients with COPD, in particular those who have frequent exacerbations and use inhaled corticosteroids. This potential signal requires confirmation in an appropriately designed prospective clinical trial. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03281876.

Subclass and avidity of circumsporozoite protein specific antibodies associate with protection status against malaria infection.

RTS,S/AS01 is an advanced pre-erythrocytic malaria vaccine candidate with demonstrated vaccine efficacy up to 86.7% in controlled human malaria infection (CHMI) studies; however, reproducible immune correlates of protection (CoP) are elusive. To identify candidates of humoral correlates of vaccine mediated protection, we measured antibody magnitude, subclass, and avidity for Plasmodium falciparum (Pf) circumsporozoite protein (CSP) by multiplex assays in two CHMI studies with varying RTS,S/AS01B vaccine dose and timing regimens. Central repeat (NANP6) IgG1 magnitude correlated best with protection status in univariate analyses and was the most predictive for protection in a multivariate model. NANP6 IgG3 magnitude, CSP IgG1 magnitude, and total serum antibody dissociation phase area-under-the-curve for NANP6, CSP, NPNA3, and N-interface binding were also associated with protection status in the regimen adjusted univariate analysis. Identification of multiple immune response features that associate with protection status, such as antibody subclasses, fine specificity and avidity reported here may accelerate development of highly efficacious vaccines against P. falciparum.

Comprehensive Data Integration Approach to Assess Immune Responses and Correlates of RTS,S/AS01-Mediated Protection From Malaria Infection in Controlled Human Malaria Infection Trials.

RTS,S/AS01 (GSK) is the world's first malaria vaccine. However, despite initial efficacy of almost 70% over the first 6 months of follow-up, efficacy waned over time. A deeper understanding of the immune features that contribute to RTS,S/AS01-mediated protection could be beneficial for further vaccine development. In two recent controlled human malaria infection (CHMI) trials of the RTS,S/AS01 vaccine in malaria-naïve adults, MAL068 and MAL071, vaccine efficacy against patent parasitemia ranged from 44% to 87% across studies and arms (each study included a standard RTS,S/AS01 arm with three vaccine doses delivered in four-week-intervals, as well as an alternative arm with a modified version of this regimen). In each trial, RTS,S/AS01 immunogenicity was interrogated using a broad range of immunological assays, assessing cellular and humoral immune parameters as well as gene expression. Here, we used a predictive modeling framework to identify immune biomarkers measured at day-of-challenge that could predict sterile protection against malaria infection. Using cross-validation on MAL068 data (either the standard RTS,S/AS01 arm alone, or across both the standard RTS,S/AS01 arm and the alternative arm), top-performing univariate models identified variables related to Fc effector functions and titer of antibodies that bind to the central repeat region (NANP6) of CSP as the most predictive variables; all NANP6-related variables consistently associated with protection. In cross-study prediction analyses of MAL071 outcomes (the standard RTS,S/AS01 arm), top-performing univariate models again identified variables related to Fc effector functions of NANP6-targeting antibodies as highly predictive. We found little benefit-with this dataset-in terms of improved prediction accuracy in bivariate models vs. univariate models. These findings await validation in children living in malaria-endemic regions, and in vaccinees administered a fourth RTS,S/AS01 dose. Our findings support a "quality as well as quantity" hypothesis for RTS,S/AS01-elicited antibodies against NANP6, implying that malaria vaccine clinical trials should assess both titer and Fc effector functions of anti-NANP6 antibodies.

Transcriptional profiles of adjuvanted hepatitis B vaccines display variable interindividual homogeneity but a shared core signature.

The current routine use of adjuvants in human vaccines provides a strong incentive to increase our understanding of how adjuvants differ in their ability to stimulate innate immunity and consequently enhance vaccine immunogenicity. Here, we evaluated gene expression profiles in cells from whole blood elicited in naive subjects receiving the hepatitis B surface antigen formulated with different adjuvants. We identified a core innate gene signature emerging 1 day after the second vaccination and that was shared by the recipients of vaccines formulated with adjuvant systems AS01B, AS01E, or AS03. This core signature associated with the magnitude of the hepatitis B surface-specific antibody response and was characterized by positive regulation of genes associated with interferon-related responses or the innate cell compartment and by negative regulation of natural killer cell-associated genes. Analysis at the individual subject level revealed that the higher immunogenicity of AS01B-adjuvanted vaccine was linked to its ability to induce this signature in most vaccinees even after the first vaccination. Therefore, our data suggest that adjuvanticity is not strictly defined by the nature of the receptors or signaling pathways it activates but by the ability of the adjuvant to consistently induce a core inflammatory signature across individuals.

A Th1/IFNγ Gene Signature Is Prognostic in the Adjuvant Setting of Resectable High-Risk Melanoma but Not in Non-Small Cell Lung Cancer.

PURPOSE: Immune components of the tumor microenvironment (TME) have been associated with disease outcome. We prospectively evaluated the association of an immune-related gene signature (GS) with clinical outcome in melanoma and non-small cell lung cancer (NSCLC) tumor samples from two phase III studies. EXPERIMENTAL DESIGN: The GS was prospectively validated using an adaptive signature design to optimize it for the sample type and technology used in phase III studies. One-third of the samples were used as "training set"; the remaining two thirds, constituting the "test set," were used for the prospective validation of the GS. RESULTS: In the melanoma training set, the expression level of eight Th1/IFNγ-related genes in tumor-positive lymph node tissue predicted the duration of disease-free survival (DFS) and overall survival (OS) in the placebo arm. This GS was prospectively and independently validated as prognostic in the test set. Building a multivariate Cox model in the test set placebo patients from clinical covariates and the GS score, an increased number of melanoma-involved lymph nodes and the GS were associated with DFS and OS. This GS was not associated with DFS in NSCLC, although expression of the Th1/IFNγ-related genes was associated with the presence of lymphocytes in tumor samples in both indications. CONCLUSIONS: These findings provide evidence that expression of Th1/IFNγ genes in the TME, as measured by this GS, is associated with clinical outcome in melanoma. This suggests that, using this GS, patients with stage IIIB/C melanoma can be classified into different risk groups.

Association of homogeneous inflamed gene signature with a better outcome in patients with metastatic melanoma treated with MAGE-A3 immunotherapeutic.

PURPOSE: This study assessed clinical activity, safety and immunogenicity of MAGE-A3 immunotherapeutic in patients with MAGE-A3-positive metastatic melanoma. PATIENTS AND METHODS: In this open-label, multicentre, uncontrolled, Phase II study (ClinicalTrials.gov NCT00896480), patients received ≤24 doses of MAGE-A3 immunotherapeutic (4-cycle schedule). At screening, two skin lesions were biopsied for MAGE-A3 expression analysis and presence/absence of a previously identified gene signature (GS) associated with favourable clinical outcome. Clinical activity was assessed in terms of clinical response, time-to-treatment failure (TTF) and progression-free survival (PFS). Adverse events (AEs) and serious AEs (SAEs) were recorded. MAGE-A3-specific immune responses were assessed. Clinical activity and immunogenicity were analysed overall and separately in patients with 2/2 (GS+/+), 1/2 (GS+/-) or 0/2 (GS-/-) biopsies presenting GS. RESULTS: Of 49 screened patients, 32 had MAGE-A3-positive tumours; 24 (8 GS+/+, 8 GS+/-, 8 GS-/-) were treated. Two complete (GS+/+ patients) and two partial responses (one GS+/+, one GS+/-) were reported; of note, one of the two complete responses was unlikely to be related to the study treatment. Median TTF and PFS were 14.8 and 7.2 months for GS+/+, 2.3 and 2.8 months for GS+/- and 2.4 and 2.9 months for GS-/- patients. Three grade 3 AEs and two SAEs unrelated to treatment were reported. All patients were seropositive for MAGE-A3 antibodies on vaccination with no differences between the different GS profiles. MAGE-A3-specific CD4+ and CD8+ T cell immunogenicity was detected; 12/16 (75.0%) of patients presented CD4+ T cell responses. CONCLUSION: Treatment with MAGE-A3 immunotherapeutic showed signs of clinical activity in GS+/+ patients. Treatment was well tolerated and immunogenic. No differences in immune responses according to GS status were observed. TRIAL REGISTRATION NUMBER: NCT00896480 (Results).

MAGE-A3 immunotherapeutic as adjuvant therapy for patients with resected, MAGE-A3-positive, stage III melanoma (DERMA): a double-blind, randomised, placebo-controlled, phase 3 trial.

BACKGROUND: Despite newly approved treatments, metastatic melanoma remains a life-threatening condition. We aimed to evaluate the efficacy of the MAGE-A3 immunotherapeutic in patients with stage IIIB or IIIC melanoma in the adjuvant setting. METHODS: DERMA was a phase 3, double-blind, randomised, placebo-controlled trial done in 31 countries and 263 centres. Eligible patients were 18 years or older and had histologically proven, completely resected, stage IIIB or IIIC, MAGE-A3-positive cutaneous melanoma with macroscopic lymph node involvement and an Eastern Cooperative Oncology Group performance score of 0 or 1. Randomisation and treatment allocation at the investigator sites were done centrally via the internet. We randomly assigned patients (2:1) to receive up to 13 intramuscular injections of recombinant MAGE-A3 with AS15 immunostimulant (MAGE-A3 immunotherapeutic; 300 µg MAGE-A3 antigen plus 420 µg CpG 7909 reconstituted in AS01B to a total volume of 0·5 mL), or placebo, over a 27-month period: five doses at 3-weekly intervals, followed by eight doses at 12-weekly intervals. The co-primary outcomes were disease-free survival in the overall population and in patients with a potentially predictive gene signature (GS-positive) identified previously and validated here via an adaptive signature design. The final analyses included all patients who had received at least one dose of study treatment; analyses for efficacy were in the as-randomised population and for safety were in the as-treated population. This trial is registered with ClinicalTrials.gov, number NCT00796445. FINDINGS: Between Dec 1, 2008, and Sept 19, 2011, 3914 patients were screened, 1391 randomly assigned, and 1345 started treatment (n=895 for MAGE-A3 and n=450 for placebo). At final analysis (data cutoff May 23, 2013), median follow-up was 28·0 months [IQR 23·3-35·5] in the MAGE-A3 group and 28·1 months [23·7-36·9] in the placebo group. Median disease-free survival was 11·0 months (95% CI 10·0-11·9) in the MAGE-A3 group and 11·2 months (8·6-14·1) in the placebo group (hazard ratio [HR] 1·01, 0·88-1·17, p=0·86). In the GS-positive population, median disease-free survival was 9·9 months (95% CI 5·7-17·6) in the MAGE-A3 group and 11·6 months (5·6-22·3) in the placebo group (HR 1·11, 0·83-1·49, p=0·48). Within the first 31 days of treatment, adverse events of grade 3 or worse were reported by 126 (14%) of 894 patients in the MAGE-A3 group and 56 (12%) of 450 patients in the placebo group, treatment-related adverse events of grade 3 or worse by 36 (4%) patients given MAGE-A3 vs six (1%) patients given placebo, and at least one serious adverse event by 14% of patients in both groups (129 patients given MAGE-A3 and 64 patients given placebo). The most common adverse events of grade 3 or worse were neoplasms (33 [4%] patients in the MAGE-A3 group vs 17 [4%] patients in the placebo group), general disorders and administration site conditions (25 [3%] for MAGE-A3 vs four [<1%] for placebo) and infections and infestations (17 [2%] for MAGE-A3 vs seven [2%] for placebo). No deaths were related to treatment. INTERPRETATION: An antigen-specific immunotherapeutic alone was not efficacious in this clinical setting. Based on these findings, development of the MAGE-A3 immunotherapeutic for use in melanoma has been stopped. FUNDING: GlaxoSmithKline Biologicals SA.

A predictive enrichment procedure to identify potential responders to a new therapy for randomized, comparative controlled clinical studies.

To evaluate a new therapy versus a control via a randomized, comparative clinical study or a series of trials, due to heterogeneity of the study patient population, a pre-specified, predictive enrichment procedure may be implemented to identify an "enrichable" subpopulation. For patients in this subpopulation, the therapy is expected to have a desirable overall risk-benefit profile. To develop and validate such a "therapy-diagnostic co-development" strategy, a three-step procedure may be conducted with three independent data sets from a series of similar studies or a single trial. At the first stage, we create various candidate scoring systems based on the baseline information of the patients via, for example, parametric models using the first data set. Each individual score reflects an anticipated average treatment difference for future patients who share similar baseline profiles. A large score indicates that these patients tend to benefit from the new therapy. At the second step, a potentially promising, enrichable subgroup is identified using the totality of evidence from these scoring systems. At the final stage, we validate such a selection via two-sample inference procedures for assessing the treatment effectiveness statistically and clinically with the third data set, the so-called holdout sample. When the study size is not large, one may combine the first two steps using a "cross-training-evaluation" process. Comprehensive numerical studies are conducted to investigate the operational characteristics of the proposed method. The entire enrichment procedure is illustrated with the data from a cardiovascular trial to evaluate a beta-blocker versus a placebo for treating chronic heart failure patients.

Predictive gene signature in MAGE-A3 antigen-specific cancer immunotherapy.

PURPOSE: To detect a pretreatment gene expression signature (GS) predictive of response to MAGE-A3 immunotherapeutic in patients with metastatic melanoma and to investigate its applicability in a different cancer setting (adjuvant therapy of resected early-stage non-small-cell lung cancer [NSCLC]). PATIENTS AND METHODS: Patients were participants in two phase II studies of the recombinant MAGE-A3 antigen combined with an immunostimulant (AS15 or AS02B). mRNA from melanoma biopsies was analyzed by microarray analysis and quantitative polymerase chain reaction. These results were used to identify and cross-validate the GS, which was then applied to the NSCLC data. RESULTS: In the patients with melanoma, 84 genes were identified whose expression was potentially associated with clinical benefit. This effect was strongest when the immunostimulant AS15 was included in the immunotherapy (hazard ratio [HR] for overall survival, 0.37; 95% CI, 0.13 to 1.05; P = .06) and was less strong with the other immunostimulant AS02B (HR, 0.84; 95% CI, 0.36 to 1.97; P = .70). The same GS was then used to predict the outcome for patients with resected NSCLC treated with MAGE-A3 plus AS02B; actively treated GS-positive patients showed a favorable disease-free interval compared with placebo-treated GS-positive patients (HR, 0.42; 95% CI, 0.17 to 1.03; P = .06), whereas among GS-negative patients, no such difference was found (HR, 1.17; 95% CI, 0.59 to 2.31; P = .65). The genes identified were mainly immune related, involving interferon gamma pathways and specific chemokines, suggesting that their pretreatment expression influences the tumor's immune microenvironment and the patient's clinical response. CONCLUSION: An 84-gene GS associated with clinical response for MAGE-A3 immunotherapeutic was identified in metastatic melanoma and confirmed in resected NSCLC.

Efficient non-viral integration and stable gene expression in multipotent adult progenitor cells.

Non-viral integrating systems, PhiC31 phage integrase (ϕC31), and Sleeping Beauty transposase (SB), provide an effective method for ex vivo gene delivery into cells. Here, we used a plasmid-encoding GFP and neomycin phosphotransferase along with recognition sequences for both ϕC31 and SB integrating systems to demonstrate that both systems effectively mediated integration in cultured human fibroblasts and in rat multipotent adult progenitor cells (rMAPC). Southern blot analysis of G418-resistant rMAPC clones showed a 2-fold higher number of SB-mediated insertions per clone compared to ϕC31. Sequence identification of chromosomal junction sites indicated a random profile for SB-mediated integrants and a more restricted profile for ϕC31 integrants. Transgenic rMAPC generated with both systems maintained their ability to differentiate into liver and endothelium albeit with marked attenuation of GFP expression. We conclude that both SB and ϕC31 are effective non-viral integrating systems for genetic engineering of MAPC in basic studies of stem cell biology.

Differentiation potential of human postnatal mesenchymal stem cells, mesoangioblasts, and multipotent adult progenitor cells reflected in their transcriptome and partially influenced by the culture conditions.

Several adherent postnatal stem cells have been described with different phenotypic and functional properties. As many of these cells are being considered for clinical therapies, it is of great importance that the identity and potency of these products is validated. We compared the phenotype and functional characteristics of human mesenchymal stem cells (hMSCs), human mesoangioblasts (hMab), and human multipotent adult progenitor cells (hMAPCs) using uniform standardized methods. Human MAPCs could be expanded significantly longer in culture. Differences in cell surface marker expression were found among the three cell populations with CD140b being a distinctive marker among the three cell types. Differentiation capacity towards adipocytes, osteoblasts, chondrocytes, and smooth muscle cells in vitro, using established protocols, was similar among the three cell types. However, only hMab differentiated to skeletal myocytes, while only hMAPCs differentiated to endothelium in vitro and in vivo. A comparative transcriptome analysis confirmed that the three cell populations are distinct and revealed gene signatures that correlated with their specific functional properties. Furthermore, we assessed whether the phenotypic, functional, and transcriptome features were mediated by the culture conditions. Human MSCs and hMab cultured under MAPC conditions became capable of generating endothelial-like cells, whereas hMab lost some of their ability to generate myotubes. By contrast, hMAPCs cultured under MSC conditions lost their endothelial differentiation capacity, whereas this was retained when cultured under Mab conditions, however, myogenic capacity was not gained under Mab conditions. These studies demonstrate that hMSCs, hMab, and hMAPCs have different properties that are partially mediated by the culture conditions.

Maintenance of HSC by Wnt5a secreting AGM-derived stromal cell line.

OBJECTIVE: The microenvironment wherein hematopoietic stem cells (HSC) reside orchestrates HSC self-renewal vs. differentiation decisions. Stromal cells derived from ontogenically divergent hematopoietic microenvironments can support HSC in vitro and have been used to decipher factors that influence HSC fate decisions. Employing stromal cell lines derived from the aorta-gonad-mesonephros and embryonic liver, we aim to identify secreted factors that maintain/expand HSC in vitro. MATERIALS AND METHODS: We cultured murine lineage antigen-negative (Lin(-)) bone marrow cells in transwells above the UG26-1B6, urogenital ridge-, and EL08-1D2, embryonic liver-derived cell lines. We, also, performed real-time quantitative PCR analysis to identify differentially expressed genes from the Wnt family of proteins in ontogenically different stromal cell lines. RESULTS: Lin(-) murine bone marrow cells maintained for 3 weeks in transwells above UG26-1B6 but not EL08-1D2 cells contained competitive repopulating HSC. Addition of as few as 25% UG26-1B6 cells to EL08-1D2 feeders led to maintenance of HSC in noncontact cultures, validating soluble factors are secreted by the UG26-1B6 cells. As we found that Wnt5a was significantly higher expressed in UG26-1B6 than EL08-1D2 cells, we added Wnt5a to EL08-1D2 transwell cultures or an antibody against Wnt5a to UG26-1B6 transwell cultures. Addition of Wnt5a to EL08-1D2 transwell cultures restored maintenance of HSC, whereas addition of an anti-Wnt5a antibody to UG26-1B6 transwell cultures inhibited maintenance of competitive repopulating HSC. CONCLUSIONS: We demonstrate that stromal cell lines generated from embryonic microenvironments provide a tool to identify secreted proteins that play a role in the maintenance of HSC, and that at least one of the factors produced by UG26-1B6 cells responsible for preserving HSC is Wnt5a.

Deficiency of either P-glycoprotein or breast cancer resistance protein protect against acute kidney injury.

The kidney has a high capacity to regenerate after ischemic injury via several mechanisms, one of which involves bone marrow-derived (stem) cells. The ATP binding cassette transporters, P-glycoprotein and breast cancer resistance protein, are determinants for the enriched stem and progenitor cell fraction in bone marrow. Because they are upregulated after acute kidney injury, we hypothesized that both efflux pumps may play a role in protecting against renal injury. Surprisingly, transporter-deficient mice were protected against ischemia-induced renal injury. To further study this, bone marrow from irradiated wild-type mice was reconstituted by bone marrow from wild-type, P-glycoprotein- or breast cancer resistance protein-deficient mice. Four weeks later, kidney injury was induced and its function evaluated. Significantly more bone marrow-derived cells were detected in kidneys grafted with transporter-deficient bone marrow. A gender mismatch study suggested that cell fusion of resident tubular cells with bone marrow cells was unlikely. Renal function analyses indicated an absence of renal damage following ischemia-reperfusion in animals transplanted with transporter-deficient bone marrow. When wild-type bone marrow was transplanted in breast cancer resistance protein-deficient mice this protection is lost. Furthermore, we demonstrate that transporter-deficient bone marrow contained significantly more monocytes, granulocytes, and early outgrowth endothelial progenitor cells.

Isolation procedure and characterization of multipotent adult progenitor cells from rat bone marrow.

Multipotent adult progenitor cells (MAPCs) are adult stem cells derived from the bone marrow of mouse and rat and were described for the first time in 2002 (Jiang et al., Nature 418:41-49, 2002), and subsequently (Breyer et al., Exp Hematol 34:1596-1601, 2006; Jiang et al., Exp Hematol 30:896-904, 2002; Ulloa-Montoya et al., Genome Biol 8:R163, 2007). The capacity of rodent MAPC to differentiate at the single-cell level into some of the cell types of endoderm, mesoderm, and neuroectoderm germ layer lineages makes them promising candidates for the study of developmental processes. MAPC are isolated using adherent cell cultures and are selected based on morphology after a period of about 8-18 weeks. Here, we describe a step-by-step reproducible method to isolate rat MAPC from fetal and adult bone marrow. We elaborate on several aspects of the isolation protocol including, cell density and medium components, and methods for selecting and obtaining potential MAPC clones and their characterization.

Self-renewal and differentiation capacity of young and aged stem cells.

Because of their ability to self-renew and differentiate, adult stem cells are the in vivo source for replacing cells lost on a daily basis in high turnover tissues during the life of an organism. Adult stem cells however, do suffer the effects of aging resulting in decreased ability to self-renew and properly differentiate. Aging is a complex process and identification of the mechanisms underlying the aging of (stem) cell population(s) requires that relatively homogenous and well characterized populations can be isolated. Evaluation of the effect of aging on one such adult stem cell population, namely the hematopoietic stem cell (HSC), which can be purified to near homogeneity, has demonstrate that they do suffer cell intrinsic age associated changes. The cells that support HSC, namely marrow stromal cells, or mesenchymal stem cells (MSC), may similarly be affected by aging, although the inability to purify these cells to homogeneity precludes definitive assessment. As HSC and MSC are being used in cell-based therapies clinically, improved insight in the effect of aging on these two stem cell populations will probably impact the selection of sources for these stem cells.

Comparative transcriptome analysis of embryonic and adult stem cells with extended and limited differentiation capacity.

BACKGROUND: Recently, several populations of postnatal stem cells, such as multipotent adult progenitor cells (MAPCs), have been described that have broader differentiation ability than classical adult stem cells. Here we compare the transcriptome of pluripotent embryonic stem cells (ESCs), MAPCs, and lineage-restricted mesenchymal stem cells (MSCs) to determine their relationship. RESULTS: Applying principal component analysis, non-negative matrix factorization and k-means clustering algorithms to the gene-expression data, we identified a unique gene-expression profile for MAPCs. Apart from the ESC-specific transcription factor Oct4 and other ESC transcripts, some of them associated with maintaining ESC pluripotency, MAPCs also express transcripts characteristic of early endoderm and mesoderm. MAPCs do not, however, express Nanog or Sox2, two other key transcription factors involved in maintaining ESC properties. This unique molecular signature was seen irrespective of the microarray platform used and was very similar for both mouse and rat MAPCs. As MSC-like cells isolated under MAPC conditions are virtually identical to MSCs, and MSCs cultured in MAPC conditions do not upregulate MAPC-expressed transcripts, the MAPC signature is cell-type specific and not merely the result of differing culture conditions. CONCLUSION: Multivariate analysis techniques clustered stem cells on the basis of their expressed gene profile, and the genes determining this clustering reflected the stem cells' differentiation potential in vitro. This comparative transcriptome analysis should significantly aid the isolation and culture of MAPCs and MAPC-like cells, and form the basis for studies to gain insights into genes that confer on these cells their greater developmental potency.

Islet-derived fibroblast-like cells are not derived via epithelial-mesenchymal transition from Pdx-1 or insulin-positive cells.

As recent studies suggest that newly formed pancreatic beta-cells are a result of self-duplication rather than stem cell differentiation, in vitro expansion of beta-cells presents a potential mechanism by which to increase available donor tissue for cell-based diabetes therapies. Although most studies have found that beta-cells are resilient to substantial in vitro expansion, recent studies have suggested that it is possible to expand these cells through a process referred to as epithelial-mesenchymal transition (EMT). To further substantiate such an expansion mechanism, we used recombination-based genetic lineage tracing to determine the origin of proliferating fibroblast-like cells from cultured pancreatic islets in vitro. We demonstrate, using two culture methods, that EMT does not underlie the appearance of fibroblast-like cells in mouse islet cultures but that fibroblast-like cells appear to represent mesenchymal stem cell (MSC)-like cells akin to MSCs isolated from bone marrow.

Culture systems for pluripotent stem cells.

Pluripotent stem cells have the capacity to self renew and to differentiate to cells of the three somatic germ layers that comprise an organism. Embryonic stem cells are the most studied pluripotent stem cells. Pluripotent stem cells have also been derived from adult tissues. Both embryonic and adult stem cells represent valuable sources of cells for applications in cell therapy, drug screening and tissue engineering. While expanding stem cells in culture, it is critical to maintain their self-renewal and differentiation capacity. In generating particular cell types for specific applications, it is important to direct their differentiation to the desired lineage. Challenges in expansion of undifferentiated stem cells for clinical applications include the removal of feeder layers and non-defined components in the culture medium. Our limited basic knowledge on the requirements for maintaining pluripotency of adult pluripotent stem cells and the lack of appropriate markers associated with pluripotency hinders the progress toward their wide spread application. In vitro differentiation of stem cells usually produces a mixed population of different cell lineages with the desired cell type present only at a small proportion. Use of growth factors that promote differentiation, expansion or survival of specific cell types is key in controlling the differentiation towards specific cell lineages. A variety of bioreactors for cell cultivation exist and can be readily adapted for stem cell cultivation and differentiation. They provide a well-controlled environment for studying the process of stem cell propagation and differentiation. Their wide use will facilitate the development of processes for stem cell application.