Human induced pluripotent stem cells (iPSCs) represent a scalable cell source for the generation of hematopoietic progenitor cells (iHPCs); however, a lack of efficient iHPC expansion in vitro currently limits translational applications. To address this translational bottleneck, we assessed a panel of stem cell agonist cocktails (SCACs), originally developed to enhance cord-blood derived HSPC (CB-HSPC) expansion, on iHPC expansion. Three SCACs and GAS6 (X2A, X2A+GAS6, SM6, or SMA) were supplemented during iHPC differentiation and subsequent expansion using the STEMdiff™ Hematopoietic Kit. This monolayer differentiation strategy yielded a population of CD34⁺CD43⁺ and CD45⁺CD34⁺ iHPC. SCAC supplementation during iHPC differentiation yielded up to 2.5-fold higher frequency of CD34⁺CD43⁺ hematopoietic progenitors and up to 2.9-fold higher frequency of CD45⁺CD34⁺CD45RA⁻CD90⁺ HSC-like cells compared to non-treated controls. Subsequent SCAC supplementation during 2 weeks of expansion culture also significantly increased iHPC expansion (X2A+GAS6: 3.8-fold, X2A: 3.5-fold, SM6: 2.8-fold, SMA: 2.0-fold). The expanded iHPCs retained high levels of CD34⁺CD43⁺ expression but we observed an increase in the expansion of HSC-like cell fraction. The collective expansion observed with the SCACs was 1.5- to 2.8-fold higher than UM171 treatment alone. Furthermore, all SCAC-supplemented iHPCs retained multilineage potency, producing erythroid and granulocyte-macrophage progenitors in CFU assays. However, prolonged expansion, beyond 7 days, reduced multilineage potential, indicating a limited expansion window. Although optimal timing and composition of SCAC supplementation remains to be refined, these results highlight that exploiting the additive and synergistic effects of multiple small molecules represents a promising approach for enhancing iHPC expansion yields and biomanufacturing.

Transforming growth factor beta 3 (TGF3) promotes tenogenic differentiation and may enhance tendon regeneration in vivo. This study aimed to apply TGF3 absorbed in decellularized equine superficial digital flexor tendon scaffolds, and to investigate the bioactivity of scaffold-associated TGF3 in an in vitro model. TGF3 could effectively be loaded onto tendon scaffolds so that at least 88% of the applied TGF3 were not detected in the rinsing fluid of the TGF3-loaded scaffolds. Equine adipose tissue-derived multipotent mesenchymal stromal cells (MSC) were then seeded on scaffolds loaded with 300 ng TGF3 to assess its bioactivity. Both scaffold-associated TGF3 and TGF3 dissolved in the cell culture medium, the latter serving as control group, promoted elongation of cell shapes and scaffold contraction (p < 0.05). Furthermore, scaffold-associated and dissolved TGF3 affected MSC musculoskeletal gene expression in a similar manner, with an upregulation of tenascin c and downregulation of other matrix molecules, most markedly decorin (p < 0.05). These results demonstrate that the bioactivity of scaold-associated TGF3 is preserved, thus TGF3 application via absorption in decellularized tendon scaffolds is a feasible approach.

As células-tronco hematopoéticas (CTHs) e estromais mesenquimais multipotentes (CTMs) isoladas da medula óssea vêm sendo utilizadas como fonte autóloga no tratamento de doenças autoimunes, como a esclerose múltipla (EM). As CTHs dão origem a todas as células dos sistemas hematopoético e imunológico e as CTMs possuem propriedades imunomoduladoras pela liberação de fatores solúveis e interação célula-célula. Existem trabalhos que sugerem que as doenças autoimunes sejam provenientes de defeitos intrínsecos nas células-tronco precursoras da medula óssea. Com o intuito de avaliar se as CTHs e CTMs de pacientes com EM possuem alterações intrínsecas, o objetivo geral deste trabalho foi avaliar o perfil de expressão gênica diferencial por microarrays de CTHs e CTMs de pacientes com EM, além de avaliar o perfil de expressão gênica de CTMs após o transplante autólogo de CTHs e a capacidade imunomoduladora in vitro das CTMs de pacientes. As CTHs e CTMs foram isoladas da medula óssea de pacientes com EM e doadores saudáveis, após consentimento informado. As CTHs foram isoladas por colunas imunomagnéticas e as CTMs foram isoladas por gradiente de densidade e submetidas à caracterização morfológica, imunofenotípica e capacidade de diferenciação em adipócitos e osteócitos. O RNA das CTHs e CTMs foi extraído e purificado e o perfil de expressão gênica foi avaliado por microarrays, utilizando hibridações em lâminas contendo 44.000 sondas. A capacidade imunomoduladora das CTMs de pacientes e controles foi avaliada por ensaios de cocultivo com linfócitos alogênicos e as citocinas foram quantificadas no sobrenadante por CBA flex e ELISA. Este estudo foi aprovado pelo comitê de ética do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto. Os resultados mostraram que as CTHs de pacientes possuem perfis de expressão gênica diferentes dos controles, com 2.722 genes diferencialmente expressos, envolvidos em vias de sinalização importantes para manutenção/proliferação das CTHs e diferenciação em linhagens específicas durante a hematopoese. Dentre essas sinalizações estão incluídas as vias da apoptose, Wnt, Notch, mTOR, PI3K/Akt e Ca/NFAT, sugerindo que as CTHs de pacientes com EM possuam alterações intrínsecas que podem estar relacionadas com a patogenia da doença autoimune. As CTMs isoladas de pacientes com EM exibiram aparência senescente e reduzida expressão de marcadores imunofenotípicos. Com relação à expressão gênica, as CTMs de pacientes possuem perfil diferente das CTMs controle, sendo detectados 618 genes diferencialmente expressos, incluindo genes relacionados à sinalização FGF, HGF, sinalização de moléculas de adesão e moléculas envolvidas nos processos de imunorregulação, como IL10, IL6, TGFB1, IFNGR1, IFNGR2 e HGF. O perfil de expressão gênica das CTMs de pacientes pós-transplante assemelhou-se ao perfil das CTMs pré-transplante. Ensaios de cocultivo de CTMs com linfócitos alogênicos mostraram que as CTMs de pacientes possuem capacidade antiproliferativa reduzida em relação às CTMs controle, e ainda, secreção reduzida de TGF- e IL-10 no sobrenadante das coculturas. Esses dados sugerem que as CTMs isoladas de pacientes com EM possuam alterações fenotípicas, transcricionais e funcionais. Embasados nesses achados, concluímos que as CTHs e as CTMs de pacientes com EM possuem alterações intrínsecas que podem estar relacionadas com a patogenia da doença. Uma vez que as CTMs sejam células com grande potencial terapêutico para controle da EM em pacientes refratários aos tratamentos convencionais, as alterações encontradas sugerem que CTMs de doadores saudáveis sejam mais adequadas em aplicações clínicas. / Bone marrow hematopoietic stem cells (HSCs) and mesenchymal stromal cells (MSCs) have been used as an autologous source to treat autoimmune diseases, such as multiple sclerosis (MS). HSC give rise to all hematopoietic and immune system cells, and MSCs exhibit immunomodulatory properties by releasing soluble factors and by cell-cell interactions. Evidence indicates that bone marrow stem cells obtained from patients with autoimmune diseases may present intrinsic defects. To assess whether or not HSC and MSC of MS patients have intrinsic defects, the main objective of this study was to evaluate the differential gene expression profiles of HSC and MSC from MS patients before and after autologous HSC transplantation, and additionally, to evaluate the in vitro immunomodulatory ability of patient MSCs. Bone marrow HSC and MSCs were isolated from MS patients and healthy donors. HSCs were isolated by immunomagnetic columns and MSCs were isolated by gradient density and cultured until the third passage. MSCs were characterized according to morphology, immunophenotypic markers and cell differentiation into adipocytes and osteocytes. HSC and MSCs mRNAs were extracted, purified, and the gene expression profile was evaluated by microarray hybridizations, using a platform containing 44.000 probes. The immunomodulatory activity of patient and control MSCs was assessed by coculture assays with allogeneic lymphocytes. Cytokines were quantified in coculture supernatants by ELISA and CBA flex. This study was approved by the Ethics Committee of the University Hospital of the School of Medicine of Ribeirão Preto. The results showed that the patient HSCs exhibited a distinctive gene expression profile when compared to healthy HSCs, yielding 2.722 differentially expressed genes, involved in essential HSC signaling pathways for maintenance, proliferation and differentiation into specific lineages during hematopoiesis. Among these signaling pathways were included, apoptosis, Wnt, Notch, mTOR, PI3K/Akt and Ca/NFAT, suggesting that patient HSCs have significant intrinsic transcriptional alterations that may be associated with MS pathogenesis. Regarding MSCs isolated from MS patients, they exhibited senescence appearance, decreased expression of immunophenotypic markers, and also exhibited a distinctive gene expression profile in relation to healthy MSCs, yielding 618 genes differentially expressed genes, included in FGF and HGF signaling pathways, adhesion molecules, and genes involved in immunoregulation processes, such as IL-10, IL-6, TGFB1, IFNGR1, IFNGR2 and HGF. Coculture assays of control or patient MSCs with allogeneic lymphocytes showed that patient cells exhibited reduced antiproliferative activity as compared with controls, and also exhibited reduced secretion of TGF- and IL-10 cytokines in coculture supernatants. These data suggest that MSCs isolated from MS patients have phenotypic, functional and transcriptional defects, highlighting genes related to MSC maintenance, adhesion and immunomodulatory effects. According to these results, we concluded that patient HSCs and MSCs have intrinsic defects that may be associated with the disease per se. Considering that MSCs exhibit great therapeutic potential to control MS patients refractory to conventional treatment, the major MSCs alterations observed in this study indicate that healthy MSCs may be more suitable for MS cell therapy.

Mesenchymal stem cells (MSCs) have demonstrated significant potential for clinical use due to their convenient isolation, lack of significant immunogenicity, lack of ethical controversy and their potential to differentiate into tissue-specific cell types. MSCs reside in almost all tissues including the adipose tissue. Adipose tissue has main advantages as wide distribution in the organism, suitable isolation and considerable amount of resident multipotent stem cells. Therefore, in this study, adipose tissue-derived mesenchymal stem cells (AT-MSCs) were isolated from BALB/c mice omentum and epididymis fat pats. During AT-MSCs maintenance and expansion in vitro, they were characterized for the expression of antigenic surface markers and for osteogenic, chondrogenic, and adipogenic differentiation potential. AT-MSCs form both sources expressed mesenchymal surface markers, CD73, and CD105 and were negative for a hematopoietic marker, CD45. The cultures derived from both adipose tissues differentiated into all three lineages. However, differences were observed in mesenchymal surface marker expression profiles as well as in the differentiation potential of AT-MSCs from different fat sources. Furthermore, AT-MSCs isolated from omentum fat depot were cultured with differentiation medium containing retinoic acid and testicular cell conditioned medium. After treatment periods, AT-MSCs showed Gdnf gene expression, this gene is a marker for Sertoli cells. The results showed that AT-MSCs from distinct fat depots have different characteristics related to stem cell surface marker expression profiles and differentiation potential. / Células-tronco mesenquimais têm demonstrado significativo potencial para aplicação terapêutica devido ao seu fácil isolamento, baixa imunogenicidade, ausência das implicações éticas e sua ampla plasticidade. Essas células estão nos mais diversos tecidos, destacando-se o tecido adiposo devido á sua ampla distribuição no organismo, conveniente obtenção e o considerável número de células-tronco mesenquimais multipotentes que podem ser isoladas desse tecido. Assim sendo, no presente estudo, células-tronco mesenquimais derivadas do tecido adiposo (AT-MSCs) foram isoladas do tecido adiposo localizado nas regiões próximas ao omento e testículos de camundongos BALB/c. Durante a manutenção e expansão das AT-MSCs in vitro, elas foram caracterizadas quanto à presença de marcadores antigênicos de superfície e potencial de diferenciação nas linhagens osteogênica, condrogênica e adipogênica. AT-MSCs de ambas as fontes expressaram os marcadores mesenquimais de superfície, CD73 e CD105, assim como foram negativas para o marcador de linhagens hematopoiéticas, CD45. Quanto ao potencial de diferenciação, os cultivos provenientes das duas origens de tecido adiposo apresentaram capacidade de diferenciar nas três linhagens acima citadas. Porém, foram observadas discretas diferenças tanto nos padrões de expressão dos marcadores mesenquimais de superfície quanto nos potenciais de diferenciação entre as AT-MSCs provenientes dos diferentes locais de deposição de gordura. Além disso, as AT-MSCs isoladas do tecido adiposo depositado em contato com o omento quando cultivadas com meios de diferenciação, contendo ácido retinóico e meio condicionado testicular demonstraram expressão do gene Gdnf o qual é reconhecidamente expresso pelas células de Sertoli. Portanto, os resultados obtidos demonstram que conforme a origem do tecido adiposo as AT-MSCs possuem diferentes características relacionadas aos marcadores de superfície assim como aos potenciais de diferenciação.

As células-tronco hematopoéticas (CTHs) e estromais mesenquimais multipotentes (CTMs) isoladas da medula óssea vêm sendo utilizadas como fonte autóloga no tratamento de doenças autoimunes, como a esclerose múltipla (EM). As CTHs dão origem a todas as células dos sistemas hematopoético e imunológico e as CTMs possuem propriedades imunomoduladoras pela liberação de fatores solúveis e interação célula-célula. Existem trabalhos que sugerem que as doenças autoimunes sejam provenientes de defeitos intrínsecos nas células-tronco precursoras da medula óssea. Com o intuito de avaliar se as CTHs e CTMs de pacientes com EM possuem alterações intrínsecas, o objetivo geral deste trabalho foi avaliar o perfil de expressão gênica diferencial por microarrays de CTHs e CTMs de pacientes com EM, além de avaliar o perfil de expressão gênica de CTMs após o transplante autólogo de CTHs e a capacidade imunomoduladora in vitro das CTMs de pacientes. As CTHs e CTMs foram isoladas da medula óssea de pacientes com EM e doadores saudáveis, após consentimento informado. As CTHs foram isoladas por colunas imunomagnéticas e as CTMs foram isoladas por gradiente de densidade e submetidas à caracterização morfológica, imunofenotípica e capacidade de diferenciação em adipócitos e osteócitos. O RNA das CTHs e CTMs foi extraído e purificado e o perfil de expressão gênica foi avaliado por microarrays, utilizando hibridações em lâminas contendo 44.000 sondas. A capacidade imunomoduladora das CTMs de pacientes e controles foi avaliada por ensaios de cocultivo com linfócitos alogênicos e as citocinas foram quantificadas no sobrenadante por CBA flex e ELISA. Este estudo foi aprovado pelo comitê de ética do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto. Os resultados mostraram que as CTHs de pacientes possuem perfis de expressão gênica diferentes dos controles, com 2.722 genes diferencialmente expressos, envolvidos em vias de sinalização importantes para manutenção/proliferação das CTHs e diferenciação em linhagens específicas durante a hematopoese. Dentre essas sinalizações estão incluídas as vias da apoptose, Wnt, Notch, mTOR, PI3K/Akt e Ca/NFAT, sugerindo que as CTHs de pacientes com EM possuam alterações intrínsecas que podem estar relacionadas com a patogenia da doença autoimune. As CTMs isoladas de pacientes com EM exibiram aparência senescente e reduzida expressão de marcadores imunofenotípicos. Com relação à expressão gênica, as CTMs de pacientes possuem perfil diferente das CTMs controle, sendo detectados 618 genes diferencialmente expressos, incluindo genes relacionados à sinalização FGF, HGF, sinalização de moléculas de adesão e moléculas envolvidas nos processos de imunorregulação, como IL10, IL6, TGFB1, IFNGR1, IFNGR2 e HGF. O perfil de expressão gênica das CTMs de pacientes pós-transplante assemelhou-se ao perfil das CTMs pré-transplante. Ensaios de cocultivo de CTMs com linfócitos alogênicos mostraram que as CTMs de pacientes possuem capacidade antiproliferativa reduzida em relação às CTMs controle, e ainda, secreção reduzida de TGF- e IL-10 no sobrenadante das coculturas. Esses dados sugerem que as CTMs isoladas de pacientes com EM possuam alterações fenotípicas, transcricionais e funcionais. Embasados nesses achados, concluímos que as CTHs e as CTMs de pacientes com EM possuem alterações intrínsecas que podem estar relacionadas com a patogenia da doença. Uma vez que as CTMs sejam células com grande potencial terapêutico para controle da EM em pacientes refratários aos tratamentos convencionais, as alterações encontradas sugerem que CTMs de doadores saudáveis sejam mais adequadas em aplicações clínicas. / Bone marrow hematopoietic stem cells (HSCs) and mesenchymal stromal cells (MSCs) have been used as an autologous source to treat autoimmune diseases, such as multiple sclerosis (MS). HSC give rise to all hematopoietic and immune system cells, and MSCs exhibit immunomodulatory properties by releasing soluble factors and by cell-cell interactions. Evidence indicates that bone marrow stem cells obtained from patients with autoimmune diseases may present intrinsic defects. To assess whether or not HSC and MSC of MS patients have intrinsic defects, the main objective of this study was to evaluate the differential gene expression profiles of HSC and MSC from MS patients before and after autologous HSC transplantation, and additionally, to evaluate the in vitro immunomodulatory ability of patient MSCs. Bone marrow HSC and MSCs were isolated from MS patients and healthy donors. HSCs were isolated by immunomagnetic columns and MSCs were isolated by gradient density and cultured until the third passage. MSCs were characterized according to morphology, immunophenotypic markers and cell differentiation into adipocytes and osteocytes. HSC and MSCs mRNAs were extracted, purified, and the gene expression profile was evaluated by microarray hybridizations, using a platform containing 44.000 probes. The immunomodulatory activity of patient and control MSCs was assessed by coculture assays with allogeneic lymphocytes. Cytokines were quantified in coculture supernatants by ELISA and CBA flex. This study was approved by the Ethics Committee of the University Hospital of the School of Medicine of Ribeirão Preto. The results showed that the patient HSCs exhibited a distinctive gene expression profile when compared to healthy HSCs, yielding 2.722 differentially expressed genes, involved in essential HSC signaling pathways for maintenance, proliferation and differentiation into specific lineages during hematopoiesis. Among these signaling pathways were included, apoptosis, Wnt, Notch, mTOR, PI3K/Akt and Ca/NFAT, suggesting that patient HSCs have significant intrinsic transcriptional alterations that may be associated with MS pathogenesis. Regarding MSCs isolated from MS patients, they exhibited senescence appearance, decreased expression of immunophenotypic markers, and also exhibited a distinctive gene expression profile in relation to healthy MSCs, yielding 618 genes differentially expressed genes, included in FGF and HGF signaling pathways, adhesion molecules, and genes involved in immunoregulation processes, such as IL-10, IL-6, TGFB1, IFNGR1, IFNGR2 and HGF. Coculture assays of control or patient MSCs with allogeneic lymphocytes showed that patient cells exhibited reduced antiproliferative activity as compared with controls, and also exhibited reduced secretion of TGF- and IL-10 cytokines in coculture supernatants. These data suggest that MSCs isolated from MS patients have phenotypic, functional and transcriptional defects, highlighting genes related to MSC maintenance, adhesion and immunomodulatory effects. According to these results, we concluded that patient HSCs and MSCs have intrinsic defects that may be associated with the disease per se. Considering that MSCs exhibit great therapeutic potential to control MS patients refractory to conventional treatment, the major MSCs alterations observed in this study indicate that healthy MSCs may be more suitable for MS cell therapy.

Indiana University-Purdue University Indianapolis (IUPUI) / In order to develop effective cures for diseases and decipher disease pathology, the need exists to cultivate a better understanding of human development. Existing studies employ the use of animal models to study and model human development and disease phenotypes but the evolutionary differences between humans and other species slightly limit the applicability of such animal models to effectively recapitulate human development. With the development of human pluripotent stem cells (hPSCs), including Human induced Pluripotent stem cells (hiPSCs) and Human Embryonic Stem cells (hESCs), human development can now be mirrored and recapitulated in vitro. These stem cells are pluripotent, that is, they possess the potential to generate any cell type of the body including muscle cells, nerve cells or blood cells. One of the major focuses of this study is to use hiPSCs to better understand and model human retinogenesis. The retina develops within the first three months of human development, hence rendering it inaccessible to investigation via traditional methods. However, with the advent of hiPSCs, retinal cells can be generated in a culture dish and the mechanisms underlying the specification of a retinal fate can be determined. Additionally, in order to use hiPSCs for successful cell replacement therapy, non-xenogeneic conditions need to be employed to allow for fruitful transplantation tests. Hence, another emphasis of this study has been to direct hiPSCs to generate retinal cells under non-xenogeneic conditions to facilitate their use for future translation purposes.

Indiana University-Purdue University Indianapolis (IUPUI) / Photoreceptors are a class of sensory neuronal cells that are deleteriously affected in many disorders and injuries of the visual system. Significant injury or loss of these cells often results in a partial or complete loss of vision. While previous studies have determined many necessary components of the gene regulatory network governing the establishment, development, and maintenance of these cells, the necessary and sufficient profile and timecourse of gene expression and/or silencing has yet to be elucidated. Arduous protocols do exist to derive photoreceptors in vitro utilizing pluripotent stem cells, but only recently have been able to yield cells that are disease- and/or patient-specific. The discovery that mammalian somatic cells can be directly reprogrammed to another terminally-differentiated cell phenotype has inspired an explosion of research demonstrating the successful genetic reprogramming of one cell type to another, a process which is typically both more timely and efficient than those used to derive the same cells from pluripotent stem cell sources. Therefore, the emphasis of this study was to establish a novel system to be used to determine a minimal transcriptional network capable of directly reprogramming mouse embryonic fibroblasts (MEFs) to rod photoreceptors. The tools, assays, and experimental design chosen and established herein were designed and characterized to facilitate this determination, and preliminary data demonstrated the utility of this approach for accomplishing this aim.

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