During embryonic development, precursor cells commit to specific cell fates in response to environmental cues through the establishment of lineage-specific gene expression programmes. Transcription factors are important downstream effectors of signalling pathways that initiate and maintain cell fate decisions. The haematopoietic transcription factor SCL (TAL-1) is an essential regulator of embryonic blood development. However, the exact stage at which SCL is required, its mechanisms of action, and its genomic targets are poorly understood. Characterising, jiow SCL functions - , during haematopoietic development will provide insights into how stern cells are specified. Using the embryonic stem cell/embryoid body (ES/EB) system to model early mouse development, we describe a critical role for SCL in mesoderm patterning. SCL is first expressed in PDGFRa+ FLK1+ mesoderm populations which contain lateral, paraxial and cardiac precursors. Through loss- and gain-of-function studies, we show that SCL drives lateral mesoderm specification and activates the haematopoietic programme in a direct DNA-binding independent manner, while actively repressing alternative mesodermal fates, specifically cardiac development, in a DNA-binding dependent manner. At a molecular level, we have identified direct genomic targets of SCL in Flk-1 + mesoderm populations. These include haematopoietic and cardiac transcription factors, cardiac-specific structural proteins, signalling proteins and general transcriptional repressors; thereby strengthening the dual function of SCL in mesoderm patterning. Finally, we have shown that the cardiac transcription factor GATA4 acts in a reciprocal manner, specifying cardiac precursors while repressing a lateral mesoderm fate. Collectively, this implicates SCL as a critical transcriptional regulator of cell fate decisions in early mesodermal precursors, employing distinct molecular mechanisms to impose a blood programme. Moreover, and extending earlier reports, we document the existence of an antagonistic cross-talk between haematopoietic and cardiac lineages during mesoderm patterning. In conclusion, this work offers a cellular and molecular platform to begin to dissect the network of genetic interactions involved in these developmental processes.

House mouse hybrid zone is a complex of subspecies Mus musculus musculus, Mus musculus domesticus and their hybrids. This hybrid zone is considered as a tension zone, maintained by balance between dispersion of individuals towards the zone center and negative selection against the hybrids. Decreased anti-parasite resistance could be one of selective factors which maintain the hybrid zone. In this thesis, I use hematological methods and skin-swelling test to compare variability in mouse health state within the house mouse hybrid zone. The skin-swelling test is a method measuring pro-inflammatory immune responsiveness. Since the commonly adopted method to perform this test does not allow clear interpretation of the test results, in this thesis I also aim to optimise the test protoco . I found that utilization of concanavalin A (ConA) is more suitable in mice than application of the commonly used phytohemaglutinin (PHA). Assessment of health state of mice by both hematological methods and skin-swelling test consistently indicates increased ability of anti-parasitic resistance in the subspecies M. m. musculus compared to subspecies M. m. domesticus. Hematological examination further shows better health state of hybrid individuals compared to parental subspecies. Our results support hybrid resistance hypothesis....

Gram-negative infection has been linked to hospital water although few studies have examined whether water systems are reservoirs of nosocomial pathogens. This study investigated longitudinal recovery of the opportunistic pathogens Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Acinetobacter baumannii from water outlets of a haematology unit and evaluated Point-Of-Use Filtration (POU-F) as a control measure. In a two-year double cross-over trial, water samples and swabs were taken weekly from 39 showers/taps on the unit. Four study phases alternated between non-filtered (Phases 1 & 3), and filtered outlets (Phases 2 & 4) using Pall AquasafeTM 14-day filters. In Phases 1 & 3; 99% of 1396 samples yielded bacterial growth, with colonies generally too numerous to count. Target species were isolated from 22% of water samples (P. aeruginosa 14%; S. maltophilia 10%) and 10% of swabs. P. aeruginosa was particularly associated with handwash stations and S. maltophilia with showers. A. baumannii was not isolated. With POU-F; 22% of 1242 samples yielded bacterial growth (mean CFU/100ml ,4.6). S. maltophilia was isolated only once from water but never from outlet swabs. PCR typing identified clusters of isolates colonizing different outlets over time but no clear association between water and patient isolates was identified. The incidence of Gram negative infections remained low throughout the study. Without POU-F, water from taps/showers represented a source of bacteria including the target species. POU-F substantially reduced the frequency and number of target species from every outlet, and merits further investigation as an intervention to protect immunocompromised patients from opportunistic pathogens.

Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) both belong to the group of mature B-cell malignancies. However, MCL is typically clinically aggressive while the clinical course of CLL varies. CLL can be divided into prognostic subgroups based on IGHV mutational status and into multiple subsets based on closely homologous (stereotyped) B-cell receptors. In paper I we investigated 31 MCL cases using high-density 250K single-nucleotide polymorphism arrays and gene expression arrays. Although most copy-number aberrations (CNAs) were previously reported in MCL, a novel deletion was identified at 20q (16%) containing the candidate tumor suppressor gene ZFP64. A high proliferation gene expression signature was associated with poor prognosis, large CNAs, 7p gains and 9q losses. Losses at 1p/8p/13q/17p were associated with increased genomic complexity. In paper II we sequenced exons 4 to 8 of the TP53 gene in 119 MCL cases. 17p copy-number status was known from previous studies or determined by real-time quantitative polymerase chain reaction. TP53 mutations were detected in 14% of cases and were strongly associated with poor survival while 17p deletions were more common (32%) but did not predict survival. In papers III and IV we applied high-resolution genomic 27K methylation arrays to 20 MCL and 39 CLL samples. In paper III MCL displayed a homogenous methylation profile without correlation with the proliferation signature whereas MCL was clearly separated from CLL. Gene ontology analysis revealed enrichment of developmental genes, in particular homeobox transcription factor genes, among targets methylated in MCL. In paper IV we compared three different stereotyped CLL subsets: #1 (IGHV unmutated), #2 (IGHV3-21) and #4 (IGHV mutated). Many genes were differentially methylated between each two subsets and immune response genes (e.g. CD80 and CD86) were enriched among genes methylated in subset #1 but not in subsets #2/#4. In summary, CNAs were frequent and not random in MCL. Specific CNAs correlated with a high proliferation gene expression signature or genomic complexity. TP53 mutations predicted short survival whereas 17p deletions did not. A high proliferation signature was not associated with differential DNA methylation in MCL, which demonstrated a homogeneous methylation pattern. In contrast, genomic methylation patterns differed between MCL and CLL and between stereotyped CLL subsets.

Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-07-04T10:50:02Z No. of bitstreams: 1 eduardosiqueiraferandes.pdf: 2252400 bytes, checksum: 56e960a38ee98a43aa5d60c466f2b6e8 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-08T14:07:24Z (GMT) No. of bitstreams: 1 eduardosiqueiraferandes.pdf: 2252400 bytes, checksum: 56e960a38ee98a43aa5d60c466f2b6e8 (MD5) / Made available in DSpace on 2017-08-08T14:07:24Z (GMT). No. of bitstreams: 1 eduardosiqueiraferandes.pdf: 2252400 bytes, checksum: 56e960a38ee98a43aa5d60c466f2b6e8 (MD5) Previous issue date: 2009-08-13 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O extrato de Ginkgo biloba (EGb) é usado no tratamento e prevenção de doenças neurodegenerativas, como antiinflamatório, no tratamento da vertigem, na perda de memória relacionada a idade. Efeitos do extrato de ginkgo na reprodução demonstraram que ele inibia a fertilização por reduzir a viabilidade de espermatozóides e degenerar o oócito. Administrado a camundongos alterou o peso de órgãos como a próstata e alterou o perfil reprodutivo desses animais – levando a uma menor taxa de prenhez e aumento das mortes embrionárias. Em ratas, o EGb demonstrou causar crescimento intra-uterino restrito, aventando-se a possibilidade do efeito ter sido causado por sua atividade estrogênica. Outros trabalhos também demonstraram efeito estrogênico do Ginkgo biloba. Sabe-se que os níveis adequados de estrogênio, progesterona e de prostaglandinas são cruciais para o transporte do concepto pela tuba uterina e sua implantação uterina, portanto existe a possibilidade da administração do EGb no início da prenhez causar alterações no desenvolvimento embrionário, sendo esse o propósito do presente trabalho. Para o estudo foram usadas 68 ratas Wistar, adultas, nulíparas, prenhes, provenientes do Biotério do Centro de Biologia da Reprodução, Universidade Federal de Juiz de Fora. As ratas foram distribuídas aleatoriamente em quatro grupos experimentais (n=17): Controle e tratados Gb 3.5, Gb 7 e Gb 14, que receberam, respectivamente, 3,5, 7 e 14mg/Kg/dia do extrato de Ginkgo biloba em 1mL de suspensão aquosa, via intragástrica, uma vez ao dia, durante os oito primeiros dias de prenhez. As ratas do grupo controle receberam pelo mesmo esquema 1mL de água destilada. As ratas foram eutanasiadas, no 15o dia de prenhez, por exsanguinação total sob anestesia. Foram avaliados: sinais clínicos indicativos de toxicidade materna – entre outros, alteração do consumo de ração e de água, alteração do peso corporal, hiper ou hipoatividade, piloereção, estereotipia, perfil bioquímico e hematológico e morte –; número de corpos lúteos; peso de órgãos maternos (ovários, fígado e rins); perfil hematológico e bioquímico maternos; peso de fetos e de placentas; número de fetos viáveis, reabsorvidos e mortos; malformações fetais em membros superiores e inferiores, fechamento de tubo neural e morfologia de face. Com exceção do perfil bioquímico materno em que houve aumento do nível de colesterol e decréscimo dos níveis de ALT, uréia e creatinina, significativamente relevantes, nos animais tratados com 7 e 14mg/Kg/dia de EGb, nenhuma outra alteração significativa foi encontrada nos parâmetros maternos ou fetais avaliados. De forma geral, o tratamento com EGb em ratas Wistar, durante o período de trânsito tubário e implantação, não causou efeito tóxico no organismo materno, tampouco induziu mortes, crescimento intrauterino retardado ou malfomações fetais. Há ainda indícios de que o EGb poderia agir de forma hepatoprotetora e nefroprotetora quando administrado a ratas Wistar durante o período de prenhez quando usado nas doses 7 e 14 mg/Kg/dia. / The Ginkgo biloba extract (EGb) is mainly used in the treatment for and in the prevention of neurodegenerative diseases. It is also used in the treatment for dizziness, age-related memory loss and as an anti-inflammatory. Effects of EGb on reproduction include fertilization inhibition through the reduction of spermatozoa viability and oocyte degeneration. When administered to male mice, EGb led to alterations in the weight of organs such as the prostate, and in the reproductive behavior of these animals (leading to lower pregnancy rates and embryo death increase). In female rats, EGb was proved to cause intra-uterine growth retardation, this effect being thought to be related to its estrogenic activity. Other studies have also shown EGb estrogenic effects. Adequate levels of estrogen, progesterone and prostaglandins are known to be very important to tubal transit and uterine implantation. Hence, there is the possibility of EGb causing embryonic development alterations when administered in the beginning of pregnancy. Investigating this possibility is the aim of this thesis. In order to achieve that, the following experimental protocol was carried out with 68 nulliparous pregnant Wistar rats. The rats were randomly distributed into four experimental groups (n=17): Control, Gb 3.5, Gb 7 and Gb 14, which were treated, respectively, with zero, 3.5, 7.0 and 14.0mg/Kg/Day of EGb diluted in 1mL of distilled water through gavage, once a day, during the first eight days of pregnancy. Rats were euthanized in the 15th day of pregnancy, through total exsanguination under anesthesia. The following parameters were assessed: clinical signs of maternal toxicity – such as feed and water intake alterations, body weight alterations, hyper or hypoactivity, piloerection, stereotypy, biochemical and hematological profile and death –; number of corpora lutea; maternal organs (ovaries, liver and kidneys) weight; maternal hematological and biochemical profiles; fetuses and placentas weight; number of viable, resorpted and dead fetuses; fetal malformations in both superior and inferior members, neural tube closure and facial morphology. Amongst the parameters evaluated, alterations were found only in the maternal hematological profile. There was significant raise in the cholesterol level and reduction in the levels of ALT, urea and creatinine for the animals treated with 7 and 14mg/Kg/day of EGb. No significant alteration was found for other maternal and fetal parameters evaluated. Hence, it seems that treatment with EGb during both tubal transit and implantation periods did not cause toxic effects to the maternal organism of Wistar rats. Neither did it induce deaths, intra-uterine growth retardation or fetal malformation. There is also evidence that EGb could present hepatoprotective and nephroprotective effects when administered to Wistar rats during pregnancy in the dosages of 7 and 14 mg/Kg/day.

Acute myeloid leukemia (AML) is a malignancy of the hematopoietic system caused by somatic mutations that accumulate in hematopoietic stem and progenitor cells. The cells are thereby transformed into leukemic stem cells (LSCs), which cannot be efficiently eliminated with the standard chemotherapy treatment. Thus, LSCs pose a risk of relapse for AML patients. There- fore, identification and characterization of LSCs is a major challenge in the field of AML research. Through next generation sequencing approaches the mutational spectrum of AML cells has been established and a continuous effort is being made to resolve the order of mutation acquisition and their functional consequences. In the subgroup of AML patients that bear a mutation in Nucleophosmin 1 (NPM1 ), a mutation in DNA-methyltransferase 3 A (DNMT3A) has been often found as a co-occurring event. Evidence suggests that this mutation arises early in leukemogene- sis and marks leukemic progenitors and stem cells. However, the functional consequences of this mutation are far from being understood. In this thesis work, I set out to unravel novel functional dependencies of the DNMT3A-mutant AML cells that can be exploited for therapeutic purposes. To nominate genes that are essential for the survival of primary AML cells, I performed a func- tional RNA interference-mediated drop out screen in 38 DNMT3A- and NPM1-mutant AML patient lines. The patients in this cohort were divided into two groups, based on the treatment outcome: an early relapse (ER) group and a long term remission (LTR) group. To nominate can- didate genes in each group, I have selected 12 screens with the highest data quality and performed a differential bioinformatic analysis. The analysis yielded 7 potential candidates, from which I initially validated three: Glucocorticoid modulatory element binding protein 1 (GMEB1), Mouse double minute 4 (MDM4) – both shared between the ER and the LTR groups – and Thioredoxin domain containing protein 9 (TXNDC9 ), which scored only in the ER group. Additional rounds of validation nominated MDM4 as the strongest candidate. To investigate the role of MDM4 in LSCs, I knocked it down in three patient samples and performed the long-term culture-initiating cell assay. However, the number of progenitor colonies that formed by the end of the assay was not enough for a statistical evaluation, probably due to the low frequency of long-term culture- initiating cells in the samples. Therefore, no conclusion regarding the functional dependency of LSCs on MDM4 could be made. However, a recent study suggested that loss of MDM4 causes cell cycle arrest and induces apoptosis in leukemic cell lines and primary cells, including progenitor populations, confirming the findings of this thesis. Nevertheless, the question about the role of MDM4 in NPM1 -mutant AML cells remains open. The NPM1 involvement in the p14Arf-MDM2- p53 pathway and the deregulation of this pathway caused by the NPM1 mutation indicate that MDM4 might poses special functions in NPM1 -mutant AML. Therefore, it should be investigated if MDM4 is a particularly suitable therapeutic target in AML with NPM1 mutation.

Einleitung: Alpakas gehören zu einer Tiergruppe, die in den vergangenen Jahren im Patientengut der Tierärzte immer häufiger anzutreffen ist. Daher ist es von großer Bedeutung, sich mit der Physiologie und Pathologie dieser Tierart zu beschäftigen. Die Versorgung der Neonaten spielt dabei eine große Rolle. Da Alpakacrias, wenn überhaupt, dann erst sehr spät, klinische Anzeichen einer Erkrankung zeigen, ist es für den Untersucher von großem Nutzen, einen Einblick in den Blutgas- und Säure-Basen-Haushalt, sowie Kenntnis von den hämatologischen und blutchemischen Parametern des Neonaten zu erhalten. Des Weiteren bietet die Blutuntersuchung ein wichtiges Hilfsmittel zur Überprüfung der Versorgung mit kolostralen Antikörpern. Ziele der Untersuchungen: Ein Ziel der Untersuchungen war die Erstellung von bisher nicht vorhandenen Referenzwerten für die venöse Blutgasanalyse, die Hämatologie und für einige blutchemische Parameter bei gesunden, lebensfrischen Alpakacrias innerhalb der ersten drei Lebenstage. Des Weiteren sollte in diesem Zusammenhang die Eignung eines mobil einsetzbaren Blutgasanalysegerätes (epoc®, Fa. Alere GmbH, Köln) für die Tierart Alpaka eruiert werden. Hierfür wurden Doppelbestimmungen der Proben mit einem etablierten stationären Blutgasanalysegerät (ABL90 FLEX®, Fa. Radiometer, Kopenhagen) durchgeführt. Tiere, Material und Methoden: In die Studie wurden 20 gesunde neugeborene Alpakacrias integriert. Die Fohlen stammten vornehmlich aus Stuten, die zur Geburtsüberwachung in die Klinik eingestallt wurden, bei anderen handelte es sich um solche, die innerhalb der ersten Lebensstunden wegen vermeintlich verzögerter Tränkeaufnahme vorgestellt worden waren, was sich aber nicht bestätigte. Alle Alpakafohlen wurden nach dem Gießener Vorsorgeschema II für neonatale Equiden klinisch untersucht. Anschließend erfolgte 3-8 Stunden p. n., 24 Stunden p. n. und 72 Stunden p. n. je eine Blutprobenentnahme. Der erste Analysezeitpunkt wurde bewusst nicht unmittelbar post natum gewählt, da die Etablierung einer stabilen Prägungsphase zwischen Muttertier und Cria nach der ersten Tränkeaufnahme abgewartet werden sollte. Die Blutentnahme erfolgte nach Reinigung und Desinfektion aus der ungestauten V. jugularis externa im Bereich des sechsten Halswirbels. Die Blutgasanalyse wurde innerhalb von 15 Minuten mit den zuvor genannten Blutgasautomaten durchgeführt. Die hämatologischen Parameter wurden mit dem pocH-100 iV (Fa. Sysmex Deutschland GmbH, Norderstedt) bestimmt, die blutchemischen Untersuchungen erfolgten mit dem FUJI DRI CHEM 3500 (Fa. Sysmex Deutschland GmbH, Norderstedt). Insgesamt wurden 55 Blutproben entnommen und analysiert. Pro Analysegerät (epoc®, ABL90 FLEX®, pocH-100 iV, FUJI DRI-CHEM 3500) wurden je 55 Messungen durchgeführt. Die statistische Auswertung erfolgte mit dem Programm IBM SPSS Statistics 22.0. Die Normalverteilung wurde mittels Shapiro-Wilk-Test überprüft. Für den Gerätevergleich (epoc®/ABL90 FLEX®) fand der Wilcoxon-Test Anwendung. Der Vergleich der Zeitpunkte erfolgte über den Friedman-Test für verbundene Stichproben. Des Weiteren wurden für die einzelnen Parameter der Median und die Perzentile, bzw. der Mittelwert und die Standardabweichung bestimmt. Die grafische Darstellung erfolgte mit Boxplots und Bland-Altman-Plots. Ergebnisse: Im Gerätevergleich konnte insgesamt auf eine gute Übereinstimmung der Messwerte geschlossen werden. Signifikante und gleichzeitig klinisch relevante Unterschiede gab es lediglich bei der Bestimmung der Sauerstoffsättigung und des Hämatokrits, was auf unterschiedliche Mess- und Berechnungsmethoden bei den Geräten zurückzuführen ist. So liegen die Hämatokritwerte beim epoc® deutlich unter denen vom ABL90 FLEX®. In diesem Zusammenhang wichtig für die Interpretation der Ergebnisse ist, dass für jedes Messgerät die individuellen Referenzbereiche berücksichtigt werden müssen. Die venöse Blutgasanalyse ergab für gesunde Crias zu Beginn des ersten Lebenstages einen pH-Wert von 7,34 – 7,40, einen Sauerstoffpartialdruck (pO2) von 21,6 – 29,2 mmHg, einen Kohlendioxidpartialdruck (pCO2) von 37,3 – 46,0 mmHg, eine Sauerstoffsättigung (sO2) von 30,5 – 48,0 %, ein aktuelles Bikarbonat (HCO3-) von 21,3 – 25,1 mmol/l, eine Standardbasenabweichung (SBE) von -3,3 – 0,2 mmol/l und einen Laktatgehalt von 1,6 – 3,4 mmol/l. Der pH Wert ähnelte im Verlauf dabei dem von Kälbern und Lämmern gleichen Alters, der pO2 war insgesamt etwas niedrig, aber konstant und ähnelte über den Messzeitraum dem von Kälbern. Es wurden bei den Crias im Vergleich zu Fohlen, Kälbern und Lämmern niedrigere pCO2-Werte festgestellt. Die Sauerstoffsättigung ähnelte der von Equidenfohlen, über den Messzeitraum fällt die Konzentration im Mittel geringfügig ab, bei den anderen Vergleichstierarten steigt sie an. Die Glukosekonzentrationen waren postnatal höher als bei anderen Haustierneonaten (3-8 h p.n.: 4,4 – 8,2; 24 h p.n.: 7,3 – 12,8; 72 h p.n.: 7,3 – 16,2 mmol/l). Laktat kann nicht, wie es beim Equidenfohlen postuliert wird, als Indikator für den Gesundheitszustand eines Alpaka-Crias genutzt werden. Hämatologisch sind die spezielle Form und die hohe Anzahl der Alpakaerythrozyten, die hohe Zahl an Leukozyten und Thrombozyten (speziell bei den Crias), sowie die hohe MCHC zu nennen. Schlussfolgerungen: Es konnten teilweise bisher fehlende Daten zur venösen Blutgasanalyse für die Beurteilung der Stoffwechsellage neugeborener Alpakacrias etabliert werden. Das mobile Blutgasgerät epoc® stellt eine für die Anwendung geeignete Alternative dar.

Burkitt lymphoma is a highly aggressive B cell non-Hodgkin's lymphoma characterised by translocation of gene on chromosome 8. This translocation is usually detected by fluorescent in-situ hybridisation (FISH) studies as part of routine diagnostic work-up and prognostication. FISH testing is commonly done with the break-apart probe (BAP). This case illustrates how this testing can be falsely negative. This patient is a young male diagnosed with Stage I low-risk Burkitt with FISH negative for translocation initially on BAP testing. Additional testing with dual FISH probe detected translocation. FISH testing using BAPs alone may be falsely negative for translocations creating a diagnostic challenge and compromising the treatment approach and assessment of prognosis.

Gram-negative infection has been linked to hospital water although few studies have examined whether water systems are reservoirs of nosocomial pathogens. This study investigated longitudinal recovery of the opportunistic pathogens Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Acinetobacter baumannii from water outlets of a haematology unit and evaluated Point-Of-Use Filtration (POU-F) as a control measure. In a two-year double cross-over trial, water samples and swabs were taken weekly from 39 showers/taps on the unit. Four study phases alternated between non-filtered (Phases 1 & 3), and filtered outlets (Phases 2 & 4) using Pall AquasafeTM 14-day filters. In Phases 1 & 3; 99% of 1396 samples yielded bacterial growth, with colonies generally too numerous to count. Target species were isolated from 22% of water samples (P. aeruginosa 14%; S. maltophilia 10%) and 10% of swabs. P. aeruginosa was particularly associated with handwash stations and S. maltophilia with showers. A. baumannii was not isolated. With POU-F; 22% of 1242 samples yielded bacterial growth (mean CFU/100ml ,4.6). S. maltophilia was isolated only once from water but never from outlet swabs. PCR typing identified clusters of isolates colonizing different outlets over time but no clear association between water and patient isolates was identified. The incidence of Gram negative infections remained low throughout the study. Without POU-F, water from taps/showers represented a source of bacteria including the target species. POU-F substantially reduced the frequency and number of target species from every outlet, and merits further investigation as an intervention to protect immunocompromised patients from opportunistic pathogens. / School of Life Sciences, University of Bradford and Pall Medical (Pall Europe Ltd)

<p dir="ltr">Hematopoietic stem cells (HSCs) originating from aorta-gonad-mesonephros (AGM) could self-renew and develop into various immune cells, such as T cells, neutrophil and natural killer (NK) cells, rendering them as a promising cell source for immunotherapy. NK cells belong to the family of the innate lymphoid cells, and are employed as one of immunotherapy to cure solid and hematological malignancies including leukemia. Neutrophils are one of the granulocytes, and they are emerging as a new therapeutic target in various cancers. Due to the lack of reliable sources for the amounts of HSCs and immune cells required for clinical infusions (~10⁹ cells/patient), it remains as a major challenge to realize their full potential in targeted cell and immunotherapy. While substantial efforts have been made to generate native cell-like HSPCs and immune cells from human pluripotent stem cells (hPSCs), intricate molecular process governing the differentiation of HSCs and immune cells remain elusive, preventing the development of robust strategies for HSC and immune cell productions.</p><p dir="ltr">In this study, we first demonstrated that critical role of temporally regulating Wnt signaling in initiating AGM-like hematopoiesis across 11 hPSC lines. By inhibiting TGFβ at the stage of aorta-like CD34+SOX17⁺ hemogenic endothelium, which led to the downregulation of Wnt signaling, we established a chemically defined, feeder-free culture system that efficiently produced robust AGM-like hematopoietic cells. Furthermore, we investigated how hypoxia affects the <i>in vitro</i> hPSC differentiation into HSPCs, which resulted in a hypoxia-enhanced HSPC differentiation platform.</p><p dir="ltr">Next, the temporal roles of transcription factors (TFs), including <i>NFIL3</i>, <i>ID2</i>,<i> </i>and <i>SPI1</i>, in regulating and promoting NK cell differentiation from hPSCs are determined. <i>NFIL3</i> and <i>SPI1</i> have been reported to influence the early stages of NK cell development, while <i>ID2</i> has an impact on the generation of NK cells throughout the early and intermediate stage. We genetically modified hPSCs with doxycycline-inducible expression of <i>NFIL3</i>, <i>ID2</i>,<i> </i>and <i>SPI1</i>, and investigated their roles in NK cell induction from hPSCs. Among these three TFs, forced expression of <i>ID2</i> yielded the highest percentage of NK cells under a chemically defined, feeder-free monolayer culture condition, demonstrating that forced expression of NK-specific TFs improves the efficiency of NK cell differentiation from hPSCs.</p><p dir="ltr">Chimeric antigen receptor (CAR) is an artificial cell receptor expressed on immune T or NK cells that has been engineered to allow T or NK cells to re-target cancer cells by exclusively binding to a cancer-specific protein. CAR engineering has significantly improved the anti-tumor efficacy of NK cell therapy, resulting in 6 FDA-approved CAR-T therapies and many other ongoing clinical trials. Recently, a chlorotoxin (CLTX)-based CAR was developed and shown to specifically bind to a variety of heterogenous glioblastoma (GBM) cell lines. To test whether CLTX-CAR could improve the anti-tumor cytotoxicity of hPSC-derived NK cells, hPSCs were engineered with CLTX-CAR for stable and homogenous CAR expression via Cas9-mediated homologous recombination. The expression of CLTX-CAR did not affect the pluripotency and NK cell differentiation potential of hPSCs, and CLTX-CAR significantly improved the cytotoxicity of hPSC-derived NK cells against GBM cells.</p><p dir="ltr">Finally, we implemented a GBM-on-a-chip microfluidic model to interrogate the tumor microenvironment (TME). Microfluidics are an emerging device for investigating cancer biology with spatiotemporal control over signaling modulators by using a small volume. The interaction between hPSC-drived neutrophils and GBM was explored in this microfluidic device. GBM TME is very complex and involves many cell types, including neurons, microglia, immune T and NK cells. In the future, microfluidic models with isogenic cell components will be designed and implemented to better model GBM TME.</p><p dir="ltr">In summary, these discoveries confirm the pivotal role of Wnt signaling in guiding hPSCs towards hematopoietic lineages, while also highlighting <i>ID2</i> as a potent enhancer of NK cell differentiation from hPSC-derived hematopoietic progenitor cells. Additionally, CAR engineering enhances the anti-tumor capabilities of hPSC-derived NK cells. Furthermore, microfluidic models are employed to interrogate GBM TME.</p>