Phenotypic and functional analysis of human fetal liver hematopoietic stem cells in culture

Steady-state hematopoiesis and hematopoietic transplantation rely on the unique potential of stem cells to undergo both self-renewal and multilineage differentiation. Fetal liver (FL) represents a promising alternative source of hematopoietic stem cells (HSCs), but limited by the total cell number obtained in a typical harvest. We reported that human FL nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cells (SRCs) could be expanded under simple stroma-free culture conditions. Here, we sought to further characterize FL HSC/SRCs phenotypically and functionally before and following culture. Unexpanded or cultured FL cell suspensions were separated into various subpopulations. These were tested for long-term culture potential and for in vivo repopulating function following transplantation into NOD/SCID mice. We found that upon culture of human FL cells, a tight association between classical stem cell phenotypes, such as CD34(+) /CD38(-) and/or side population, and NOD/SCID repopulating function was lost, as observed with other sources. Although SRC activity before and following culture consistently correlated with the presence of a CD34(+) cell population, we provide evidence that, contrary to umbilical cord blood and adult sources, stem cells present in both CD34(+) and CD34(-) FL populations can sustain long-term hematopoietic cultures. Furthermore, upon additional culture, CD34-depleted cell suspensions, devoid of SRCs, regenerated a population of CD34(+) cells possessing SRC function. Our studies suggest that compared to neonatal and adult sources, the phenotypical characteristics of putative human FL HSCs may be less strictly defined, and reinforce the accumulated evidence that human FL represents a unique, valuable alternative and highly proliferative source of HSCs for clinical applications.

Identification of markers to characterize and sort human articular chondrocytes with enhanced in vitro chondrogenic capacity

OBJECTIVE: To identify markers associated with the chondrogenic capacity of expanded human articular chondrocytes and to use these markers for sorting of more highly chondrogenic subpopulations. METHODS: The chondrogenic capacity of chondrocyte populations derived from different donors (n = 21) or different clonal strains from the same cartilage biopsy specimen (n = 21) was defined based on the glycosaminoglycan (GAG) content of tissues generated using a pellet culture model. Selected cell populations were analyzed by microarray and flow cytometry. In some experiments, cells were sorted using antibodies against molecules found to be associated with differential chondrogenic capacity and again assessed in pellet cultures. RESULTS: Significance Analysis of Microarrays indicated that chondrocytes with low chondrogenic capacity expressed higher levels of insulin-like growth factor 1 and of catabolic genes (e.g., matrix metalloproteinase 2, aggrecanase 2), while chondrocytes with high chondrogenic capacity expressed higher levels of genes involved in cell-cell or cell-matrix interactions (e.g., CD49c, CD49f). Flow cytometry analysis showed that CD44, CD151, and CD49c were expressed at significantly higher levels in chondrocytes with higher chondrogenic capacity. Flow cytometry analysis of clonal chondrocyte strains indicated that CD44 and CD151 could also identify more chondrogenic clones. Chondrocytes sorted for brighter CD49c or CD44 signal expression produced tissues with higher levels of GAG per DNA (up to 1.4-fold) and type II collagen messenger RNA (up to 3.4-fold) than did unsorted cells. CONCLUSION: We identified markers that allow characterization of the capacity of monolayer-expanded chondrocytes to form in vitro cartilaginous tissue and enable enrichment for subpopulations with higher chondrogenic capacity. These markers might be used as a means to predict and possibly improve the outcome of cell-based cartilage repair techniques.

Quantitative isolation of mouse and human intestinal intraepithelial lymphocytes by elutriation centrifugation

Intestinal intraepithelial lymphocytes (IEL) are specialized subsets of T cells with distinct functional capacities. While some IEL subsets are circulating, others such as CD8alphaalpha TCRalphabeta IEL are believed to represent non-circulating resident T cell subsets [Sim, G.K., Intraepithelial lymphocytes and the immune system. Adv. Immunol., 1995. 58: 297-343.]. Current methods to obtain enriched preparations of intraepithelial lymphocytes are mostly based on Percoll density gradient or magnetic bead-based technologies [Lundqvist, C., et al., Isolation of functionally active intraepithelial lymphocytes and enterocytes from human small and large intestine. J. Immunol. Methods, 1992. 152(2): 253-263.]. However, these techniques are hampered by a generally low yield of isolated cells, and potential artifacts due to the interference of the isolation procedure with subsequent functional assays, in particular, when antibodies against cell surface markers are required. Here we describe a new method for obtaining relatively pure populations of intestinal IEL (55-75%) at a high yield (>85%) by elutriation centrifugation. This technique is equally suited for the isolation and enrichment of intraepithelial lymphocytes of both mouse and human origin. Time requirements for fractionating cell suspensions by elutriation centrifugation are comparable to Percoll-, or MACS-based isolation procedures. Hence, the substantially higher yield and the consistent robust enrichment for intraepithelial lymphocytes, together with the gentle treatment of the cells during elutriation that does not interfere with subsequent functional assays, are important aspects that are in favor of using this elegant technology to obtain unmanipulated, unbiased populations of intestinal intraepithelial lymphocytes, and, if desired, also of pure epithelial cells.

Cortical local and long-range synchronization interplay in human absence seizure initiation

Brain activity relies on transient, fluctuating interactions between segregated neuronal populations. Synchronization within a single and between distributed neuronal clusters reflects the dynamics of these cooperative patterns. Thus absence epilepsy can be used as a model for integrated, large-scale investigation of the emergence of pathological collective dynamics in the brain. Indeed, spike-wave discharges (SWD) of an absence seizure are thought to reflect abnormal cortical hypersynchronization. In this paper, we address two questions: how and where do SWD arise in the human brain? Therefore, we explored the spatio-temporal dynamics of interactions within and between widely distributed cortical sites using magneto-encephalographic recordings of spontaneous absence seizures. We then extracted, from their time-frequency analysis, local synchronization of cortical sources and long-range synchronization linking distant sites. Our analyses revealed a reproducible sequence of 1) long-range desynchronization, 2) increased local synchronization and 3) increased long-range synchronization. Although both local and long-range synchronization displayed different spatio-temporal profiles, their cortical projection within an initiation time window overlap and reveal a multifocal fronto-central network. These observations contradict the classical view of sudden generalized synchronous activities in absence epilepsy. Furthermore, they suggest that brain states transition may rely on multi-scale processes involving both local and distant interactions.

Isolation of myeloid dendritic cells and epithelial cells from human thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c(+)), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45(hi)) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

Association of PPARG2 Pro12Ala variant with larger body mass index in Mestizo and Amerindian populations of Mexico.

Previous studies have sought to associate the Pro12Ala variant of the peroxisome proliferator-activated receptor gamma2 (PPARG2) gene with type 2 diabetes, insulin resistance, and obesity, with controversial results. We have determined the Pro12Ala variant frequency in 370 nondiabetic Mexican Mestizo subjects and in five Mexican Amerindian groups and have investigated its possible association with lipid metabolism, insulin serum levels, and obesity in three of these populations. Two independent case-control studies were conducted in 239 nondiabetic individuals: 135 case subjects (BMI > or = 25 kg/m2) and 104 control subjects (BMI < 25 kg/m2). The PPARG2 Ala12 allele frequency was higher in most Amerindian populations (0.17 in Yaquis, 0.16 in Mazahuas, 0.16 in Mayans, and 0.20 in Triquis) than in Asians, African Americans, and Caucasians. The Pro12Ala and Ala12Ala (X12Ala) genotypes were significantly associated with greater BMI in Mexican Mestizos and in two Amerindian groups. X12Ala individuals had a higher risk of overweight or obesity than noncarriers in Mestizos (OR = 3.67; 95% CI, 1.42-9.48; p = 0.007) and in Yaquis plus Mazahuas (OR = 3.21; 95% CI, 1.27-8.11; p = 0.013). Our results provide further support of the association between the PPARG2 Ala12 allele and risk of overweight or obesity in Mestizos and two Amerindian populations from Mexico.

3D scaffolds co-seeded with human endothelial progenitor and mesenchymal stem cells: evidence of prevascularisation within 7 days

Blood supply is a critical issue in most tissue engineering approaches for large defect healing. As vessel ingrowth from surrounding tissues is proven to be insufficient, current strategies are focusing on the neo-vascularisation process. In the present study, we developed an in vitro pre-vascularised construct using 3D polyurethane (PU) scaffolds, based on the association of human Endothelial Progenitor Cells (EPC, CD34+ and CD133+) with human Mesenchymal Stem Cells (MSC). We showed the formation of luminal tubular structures in the co-seeded scaffolds as early as day 7 in culture. These tubular structures were proven positive for endothelial markers von Willebrand Factor and PECAM-1. Of special significance in our constructs is the presence of CD146-positive cells, as a part of the neovasculature scaffolding. These cells, coming from the mesenchymal stem cells population (MSC or EPC-depleted MSC), also expressed other markers of pericyte cells (NG2 and αSMA) that are known to play a pivotal function in the stabilisation of newly formed pre-vascular networks. In parallel, in co-cultures, osteogenic differentiation of MSCs occurred earlier when compared to MSCs monocultures, suggesting the close cooperation between the two cell populations. The presence of angiogenic factors (from autologous platelet lysates) in association with osteogenic factors seems to be crucial for both cell populations' cooperation. These results are promising for future clinical applications, as all components (cells, growth factors) can be prepared in an autologous way.

Identification and isolation of small CD44-negative mesenchymal stem/progenitor cells from human bone marrow using elutriation and polychromatic flow cytometry

The method of isolation of bone marrow (BM) mesenchymal stem/stromal cells (MSCs) is a limiting factor in their study and therapeutic use. MSCs are typically expanded from BM cells selected on the basis of their adherence to plastic, which results in a heterogeneous population of cells. Prospective identification of the antigenic profile of the MSC population(s) in BM that gives rise to cells with MSC activity in vitro would allow the preparation of very pure populations of MSCs for research or clinical use. To address this issue, we used polychromatic flow cytometry and counterflow centrifugal elutriation to identify a phenotypically distinct population of mesenchymal stem/progenitor cells (MSPCs) within human BM. The MSPC activity resided within a population of rare, small CD45⁻CD73⁺CD90⁺CD105⁺ cells that lack CD44, an antigen that is highly expressed on culture-expanded MSCs. In culture, these MSPCs adhere to plastic, rapidly proliferate, and acquire CD44 expression. They form colony forming units-fibroblast and are able to differentiate into osteoblasts, chondrocytes, and adipocytes under defined in vitro conditions. Their acquired expression of CD44 can be partially downregulated by treatment with recombinant human granulocyte-colony stimulating factor, a response not found in BM-MSCs derived from conventional plastic adherence methods. These observations indicate that MSPCs within human BM are rare, small CD45⁻CD73⁺CD90⁺CD105⁺ cells that lack expression of CD44. These MSPCs give rise to MSCs that have phenotypic and functional properties that are distinct from those of BM-MSCs purified by plastic adherence.

Camel Streptococcus agalactiae populations are associated with specific disease complexes and acquired the tetracycline resistance gene tetM via a Tn916-like element

Camels are the most valuable livestock species in the Horn of Africa and play a pivotal role in the nutritional sustainability for millions of people. Their health status is therefore of utmost importance for the people living in this region. Streptococcus agalactiae, a Group B Streptococcus (GBS), is an important camel pathogen. Here we present the first epidemiological study based on genetic and phenotypic data from African camel derived GBS. Ninety-two GBS were characterized using multilocus sequence typing (MLST), capsular polysaccharide typing and in vitro antimicrobial susceptibility testing. We analysed the GBS using Bayesian linkage, phylogenetic and minimum spanning tree analyses and compared them with human GBS from East Africa in order to investigate the level of genetic exchange between GBS populations in the region. Camel GBS sequence types (STs) were distinct from other STs reported so far. We mapped specific STs and capsular types to major disease complexes caused by GBS. Widespread resistance (34%) to tetracycline was associated with acquisition of the tetM gene that is carried on a Tn916-like element, and observed primarily among GBS isolated from mastitis. The presence of tetM within different MLST clades suggests acquisition on multiple occasions. Wound infections and mastitis in camels associated with GBS are widespread and should ideally be treated with antimicrobials other than tetracycline in East Africa.

Differences in primary sites of infection between zoonotic and human tuberculosis: results from a worldwide systematic review.

Tuberculosis (TB) is one of the most devastating infectious diseases worldwide. Whilst global burden estimates for M. tuberculosis infection (MtTB) are well established, accurate data on the contribution of zoonotic TB (zTB) caused by M. bovis or M. caprae to human TB are scarce. The association of M. bovis infection with extrapulmonary tuberculosis has been suggested repeatedly, though there is little scientific evidence available to support this relationship. The present study aimed to determine globally the occurrence of extrapulmonary TB and the primary site (i.e. primary body location affected) of zTB in comparison with MtTB, based on previously published reports. A systematic literature review was conducted in 32 different bibliographic databases, selecting reports on zTB written in English, French, German, Spanish or Portuguese. Data from 27 reports from Africa, America, Europe and the Western Pacific Region were extracted for analyses. Low income countries, in Africa and South-East Asia, were highly underrepresented in the dataset. The median proportion of extrapulmonary TB cases was significantly increased among zTB in comparison with data from registries of Europe and USA, reporting mainly MtTB cases (47% versus 22% in Europe, 73% versus 30% in the USA). These findings were confirmed by analyses of eight studies reporting on the proportions of extrapulmonary TB in comparable populations of zTB and MtTB cases (median 63% versus 22%). Also, disparities of primary sites of extrapulmonary TB between zTB and MtTB were detected. Our findings, based on global data, confirm the widely suggested association between zTB and extrapulmonary disease. Different disability weights for zTB and MtTB should be considered and we recommend separate burden estimates for the two diseases.

Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells

Human pluripotent stem cells are a powerful tool for modeling brain development and disease. The human cortex is composed of two major neuronal populations: projection neurons and local interneurons. Cortical interneurons comprise a diverse class of cell types expressing the neurotransmitter GABA. Dysfunction of cortical interneurons has been implicated in neuropsychiatric diseases, including schizophrenia, autism, and epilepsy. Here, we demonstrate the highly efficient derivation of human cortical interneurons in an NKX2.1::GFP human embryonic stem cell reporter line. Manipulating the timing of SHH activation yields three distinct GFP+ populations with specific transcriptional profiles, neurotransmitter phenotypes, and migratory behaviors. Further differentiation in a murine cortical environment yields parvalbumin- and somatostatin-expressing neurons that exhibit synaptic inputs and electrophysiological properties of cortical interneurons. Our study defines the signals sufficient for modeling human ventral forebrain development in vitro and lays the foundation for studying cortical interneuron involvement in human disease pathology.

Drug metabolism in human brain: high levels of cytochrome P4503A43 in brain and metabolism of anti-anxiety drug alprazolam to its active metabolite.

Cytochrome P450 (P450) is a super-family of drug metabolizing enzymes. P450 enzymes have dual function; they can metabolize drugs to pharmacologically inactive metabolites facilitating their excretion or biotransform them to pharmacologically active metabolites which may have longer half-life than the parent drug. The variable pharmacological response to psychoactive drugs typically seen in population groups is often not accountable by considering dissimilarities in hepatic metabolism. Metabolism in brain specific nuclei may play a role in pharmacological modulation of drugs acting on the CNS and help explain some of the diverse response to these drugs seen in patient population. P450 enzymes are also present in brain where drug metabolism can take place and modify therapeutic action of drugs at the site of action. We have earlier demonstrated an intrinsic difference in the biotransformation of alprazolam (ALP) in brain and liver, relatively more alpha-hydroxy alprazolam (alpha-OHALP) is formed in brain as compared to liver. In the present study we show that recombinant CYP3A43 metabolizes ALP to both alpha-OHALP and 4-hydroxy alprazolam (4-OHALP) while CYP3A4 metabolizes ALP predominantly to its inactive metabolite, 4-OHALP. The expression of CYP3A43 mRNA in human brain samples correlates with formation of relatively higher levels of alpha-OH ALP indicating that individuals who express higher levels of CYP3A43 in the brain would generate larger amounts of alpha-OHALP. Further, the expression of CYP3A43 was relatively higher in brain as compared to liver across different ethnic populations. Since CYP3A enzymes play a prominent role in the metabolism of drugs, the higher expression of CYP3A43 would generate metabolite profile of drugs differentially in human brain and thus impact the pharmacodynamics of psychoactive drugs at the site of action.

Human Trafficking Victims and Their Children: Assessing Needs, Vulnerabilities, Strengths, and Survivorship

Given the increased awareness and attention to human trafficking, including the establishment of federal laws and policies, federally funded task forces that provide law enforcement responses, and specialized victim services, it is important to assess the impact of these procedures and services on survivors/victims of international human trafficking and their immigrant children. By federal definition, certified victims of international human trafficking are eligible for all services provided to refugees in this country, including reunification with their minor children. This research is based on a qualitative study conducted in Austin and Houston, Texas with human trafficking victims/survivors. The project’s goal was to gain an understanding of the needs of human trafficking survivors after their rescue, their overall integration into American life, and the subsequent needs of their immigrant children after reunification. The project objectives examined the factors that either promote or hinder self-sufficiency, the determination of social service needs, and policy and practice recommendations to strengthen survivors, their children and their families living both locally and abroad. For this project, nine (n = 9) in-depth interviews were conducted with adult foreign-born victims of human trafficking. Researchers gathered data using a semi-structured questionnaire that queried about factors that promote or hinder victims’ services and needs. Interviews were conducted in participants’ homes using bilingual research staff and/or trained interpreters, were digitally-recorded, and subsequently transcribed. Participation in this study was completely voluntary. Specific steps were taken to ensure that the participants’ identities were protected. Open coding of data was utilized and the data were subsequently organized or grouped into properties and later developed into contextual themes around the research questions. The findings are grounded with the use of direct quotes from participants. As a result of progressive U.S. policy, many victims of human trafficking are being reunited with their minor children. Immigrant children are one of the largest and fastest growing populations in the U.S. and for a variety of reasons are vulnerable to exploitation. Research also indicates that victims of trafficking are identified by traffickers because of their perceived “vulnerabilities” or lack of opportunities (Clark, 2003). Therefore, it is important that practices and policies are developed to address the unique needs of these families with an eye toward positive outcomes for parent and child safety and well-being. Social service providers are provided a toolkit that may be utilized before and during the reunification period.

Drug metabolism in human brain: high levels of cytochrome P4503A43 in brain and metabolism of anti-anxiety drug alprazolam to its active metabolite.

Cytochrome P450 (P450) is a super-family of drug metabolizing enzymes. P450 enzymes have dual function; they can metabolize drugs to pharmacologically inactive metabolites facilitating their excretion or biotransform them to pharmacologically active metabolites which may have longer half-life than the parent drug. The variable pharmacological response to psychoactive drugs typically seen in population groups is often not accountable by considering dissimilarities in hepatic metabolism. Metabolism in brain specific nuclei may play a role in pharmacological modulation of drugs acting on the CNS and help explain some of the diverse response to these drugs seen in patient population. P450 enzymes are also present in brain where drug metabolism can take place and modify therapeutic action of drugs at the site of action. We have earlier demonstrated an intrinsic difference in the biotransformation of alprazolam (ALP) in brain and liver, relatively more alpha-hydroxy alprazolam (alpha-OHALP) is formed in brain as compared to liver. In the present study we show that recombinant CYP3A43 metabolizes ALP to both alpha-OHALP and 4-hydroxy alprazolam (4-OHALP) while CYP3A4 metabolizes ALP predominantly to its inactive metabolite, 4-OHALP. The expression of CYP3A43 mRNA in human brain samples correlates with formation of relatively higher levels of alpha-OH ALP indicating that individuals who express higher levels of CYP3A43 in the brain would generate larger amounts of alpha-OHALP. Further, the expression of CYP3A43 was relatively higher in brain as compared to liver across different ethnic populations. Since CYP3A enzymes play a prominent role in the metabolism of drugs, the higher expression of CYP3A43 would generate metabolite profile of drugs differentially in human brain and thus impact the pharmacodynamics of psychoactive drugs at the site of action.

Distribution of genes encoding MSCRAMMs and Pili in clinical and natural populations of Enterococcus faecium.

Enterococcus faecium has recently emerged as an important cause of nosocomial infections. We previously identified 15 predicted surface proteins with characteristics of MSCRAMMs and/or pili and demonstrated that their genes were frequently present in 30 clinical E. faecium isolates studied; one of these, acm, has been studied in further detail. To determine the prevalence of the other 14 genes among various E. faecium populations, we have now assessed 433 E. faecium isolates, including 264 isolates from human clinical infections, 69 isolates from stools of hospitalized patients, 70 isolates from stools of community volunteers, and 30 isolates from animal-related sources. A variable distribution of the 14 genes was detected, with their presence ranging from 51% to 98% of isolates. While 81% of clinical isolates carried 13 or 14 of the 14 genes tested, none of the community group isolates and only 13% of animal isolates carried 13 or 14 genes. The presence of these genes was most frequent in endocarditis isolates, with 11 genes present in all isolates, followed by isolates from other clinical sources. The number of genes significantly associated with clinical versus fecal or animal origin (P = 0.04 to <0.0001) varied from 10 to 13, depending on whether comparisons were made against individual clinical subgroups (endocarditis, blood, and other clinical isolates) or against all clinical isolates combined as one group. The strong association of these genes with clinical isolates raises the possibility that their preservation/acquisition has favored the adaptation of E. faecium to nosocomial environments and/or patients.