As células-tronco de pluripotência induzida (iPSC) representam uma fonte de maior disponibilidade para obtenção de células estromais mesenquimais (MSC). Além disso, as células mesenquimais derivadas de iPSC, as MSC-like, demonstram in vitro uma atividade parácrina e uma taxa de proliferação que as apontam como uma candidata em potencial à terapia celular. Em paralelo, a terapia celular dispõe das propriedades terapêuticas das células estromais mesenquimais do cordão umbilical (UC-MSC), uma população celular de fácil isolamento, rendimento notável, alta capacidade de proliferação e ausência de tumorigenecidade. Assim, perante a relevância das iPSC como fonte alternativa para obtenção de MSC e do desempenho in vitro demonstrado pelas MSC-like, tornou-se necessário a comparação destas com as UC-MSC. Para tanto, iPSC reprogramadas a partir de células mononucleares do sangue periférico (iPS-PBMC) foram diferenciadas em MSC-like e estas por sua vez foram caracterizadas, segundo à aderência ao plástico, expressão de genes e proteínas de pluripotência, expressão de antígenos de superfície CD73, CD90, CD105, CD14, CD19, CD34, CD45 e HLA-DR; capacidade de diferenciação in vitro em adipócitos e osteócitos e posteriormente comparadas as UC-MSC frente a capacidade de proliferação e imunomodulação. As MSC-like obtidas mostraram-se aderentes ao plástico, não pluripotentes e com morfologia fibroblastoide. Demonstraram um perfil imunofenotípico negativo (menos de 2% de células positivas) para marcadores hematopoéticos e HLA-DR, mais de 90% para CD73 e CD90 e menos de 80% para CD105. Ademais, exibiram capacidade de diferenciação osteogênica e adipogênica. Quando comparadas às UC-MSC, em relação à capacidade de proliferação, as MSC-like apresentaram uma taxa de proliferação duas vezes menor. A comparação da capacidade imunomodulatória entre as duas linhagens demonstrou que as UC-MSC foram capazes de conter a proliferação de linfócitos T CD8+ mas não de linfócitos T CD4+. As MSC-like não foram capazes de conter a proliferação de ambas as populações de linfócitos. Os resultados indicam que o protocolo de indução utilizado foi capaz de gerar MSC-like com antígenos de superfície clássico em mesenquimais, baixa expressão de marcadores de pluripotência, hematopoéticos e HLA-DR e com habilidade de se diferenciar em linhagens mesodérmicas, porém, com menor capacidade de proliferação e ausência de propriedades imunomodulatórias. A funcionalidade das MSC-like geradas neste trabalho pode ser proveniente de fatores genéticos e epigenéticos do doador e pelas metodologias de reprogramação da célula somática à iPSC, bem como pela indução da iPSC à MSC. A investigação futura destes fatores pode contribuir para a obtenção de MSC-like funcionalmente semelhante às UC-MSC. / Induced pluripotent stem cells (iPSC) are an easily available mesenchymal stromal cell (MSC) source. Moreover, iPSC-derived mesenchymal cells (MSC-like) demonstrated an in vitro proliferation rate and paracrine activity that make them a potential candidate for cell therapy. Currently the cell therapy uses the therapeutic properties of umbilical cord-derived mesenchymal cells (UC-MSC), an effortlessly isolated cell population with remarkable yield, high proliferation capacity, and absence of tumorigenecity. Thus, considering the iPSC relevance as an alternative MSC source and their in vitro performance, it has become necessary to compare them with UC-MSC. The iPSC reprogrammed from peripheral blood mononuclear cells (iPS-PBMC) were differentiated in MSC-like and these cells were characterized according their adherence to plastic; pluripotency genes and proteins expression; surface antigens expression such CD73, CD90, CD105, CD14, CD19, CD34, CD45, and HLA-DR; in vitro differentiation capacity into adipocytes and osteocytes; and were compared with the UC-MSC proliferation and immunomodulation capacity. The obtained MSC-like were adherent to plastic, not pluripotent, and with fibroblastoid morphology. They demonstrated an immunophenotypic profile with less than 2% hematopoietic and HLA-DR positive cell markers, over 90% of CD73 and CD90, and less than 80% were CD105 positives. They also exhibited osteogenic and adipogenic differentiation capacity. When proliferation rate was compared between the two cell lineages, MSC-like showed a proliferation rate twice smaller than UC-MSC. The UC-MSC immunomodulatory activity contained the proliferation of T CD8+ lymphocytes but did not of T CD4+. MSC-like did not show immunomodulatory activity in the proliferation of CD4+ and CD8+. These results allow the assumption that the applied induction protocol was able to generate MSC-like with the classic mesenchymal surface markers, low pluripotency, hematopoietic, and HLA-DR surface markers expression and with mesodermal lineage differentiation potential, however, with lower proliferation rate and absence of immunomodulatory properties. The MSC-like functionality could be influenced by donor genetic and epigenetic factors as well as by as well as the methodologies of reprogramming of somatic cells to the iPSC and the induction of iPSC to the mesenchymal phenotype. These influential factors should be investigated in deep detail in order to make the generated MSC-like functionally similar to UC-MSC.

Cellers tillväxthastighet varierar avsevärt mellan olika celltyper. Fullt specialiserade celler delar sig sällan och fokuserar i stället på sina specifika funktioner, medan stamceller aktivt delar sig för att upprätthålla en balans av nya och gamla celler. Denna studie syftar till att odla och analysera två mänskliga blodcancercellinjer: erytroleukemiska K562-celler och L428-celler från Hodgkins lymfom. Studien fokuserar på de två cellinjernas olika tillväxthastighet samt transkriptionsaktivitet under olika förhållanden och behandlingar. Projektet inkluderar även att ta fram ett helt nytt, effektiviserat protokoll för extraktion och kvantifiering av DNA, nascent RNA, mRNA och protein från ett enda prov. Projektet tar också upp utmaningen att se om det finns en koppling mellan cellernas tillstånd och deras transkriptionsaktivitet, eftersom nuvarande sekvenseringsnormaliseringstekniker gör att de totala nivåerna av transkription inte blir jämförbara mellan olika cellinjer. Genom att kvantifiera cellernas tillväxthastighet och transkriptionsaktivitet identifierades en potentiell koppling mellan högre tillväxthastighet och ökad mängd RNA-produktion, vilket tyder på ett samband som ser till att tillräckligt stor mängd cellkomponenter produceras för dottercellerna. Dessutom syftar studien till att undersöka de två cellinjernas respons på behandling med differentieringsinducerande hemin, känt för att inducera erytroid differentiering hos myeloida celler såsom K562-cellinjen. Trots deras olika hematopoetiska ursprung visade sig både K562- och L428-cellinjerna reagera på behandlingen och utvecklas mot röda blodkroppar. Detta kan påvisas genom att hemin gör att deras respektive tillväxthastigheter påverkas på samma sätt, samt att cellerna får en djupröd färg, vilket indikerar produktion av hemoglobin och är ett välkänt tecken på erytroid differentiering. / Cell proliferation rates vary significantly among different cell types. Terminally differentiated cells rarely divide, focusing on their specialized functions, while stem cells actively proliferate to maintain tissue homeostasis. This study aims to culture and analyze two human blood cancer cell lines: erythroleukemia K562 cells and Hodgkin's lymphoma L428 cells. The investigation focuses on their proliferation rates and transcriptional activities under various conditions and treatments. The project also includes the establishment of a novel, streamline protocol for extracting and quantifying DNA, nascent RNA, mRNA and protein from one single sample. The study also addresses the challenge of correlating cell state with transcriptional activity, noting that current sequencing normalization techniques renders total levels of transcription incomparable between cell lines. By quantifying proliferation rates and transcriptional activity, a potential connection between proliferation rate and transcriptional activity was found. Higher proliferation rate corresponded to samples with larger amounts of RNA and higher transcription of nascent RNA, indicating an association that facilitates the production of sufficient cellular components necessary for the two daughter cells.  Additionally, the study investigates the two cell lines’ responsiveness to differentiation-inducing hemin, known to induce differentiation toward the erythrocyte lineage for myeloid cells such as the K562 cell line. The findings from this study show that, despite belonging to different parts of the hematopoietic lineage, both the K562 cells and the L428 cells are responsive to hemin-induced differentiation toward the erythrocyte lineage. This is demonstrated by the fact that their proliferation rates are equally affected upon hemin treatments, and because both K562 and L428 cells turn red as a reflection of the induced production of hemoglobin, which is a recognized effect of hemin-induced erythrocytic differentiation.