Gostaríamos de agradecer o apoio da Universidade de Chicago Seção de Urologia liderada pelo Dr. Arieh Shalhav, eo Diretor de Pesquisa Urológica Dr. Carrie Rinker-Schaeffer. Gostaríamos também de agradecer o apoio da Universidade de Chicago Comprehensive Cancer Center (UCCCC) liderada pelo Dr. Michelle Le Beau. Contamos também com a agradecer a assistência técnica especializada da Tissue Resource Center facilidade núcleo Human liderada pelo Dr. Mark Lingen, ea assistência de Leslie Martin e Mary Jo Fekete. Agradecemos também a Facilidade Núcleo Imunohistoquímica dirigido por Terri Li. Este trabalho foi financiado pela Universidade de Chicago Departamento de Cirurgia, da Seção de Urologia; uma Sociedade Americana do Câncer Grant Pesquisa Institucional (ACS-IRG, # IRG-58-004), uma Cancer Support Center Grant (P30 CA14599); A Brinson Foundation, o Fund Alvin Baum Família; The University of Chicago Board Cancer Research Foundation da Mulher; S. Kregel é apoiado por um HHMI: Med-em-Grad Fellowsquadril (56006772) e Biologia do Câncer Training Grant (T32-CA09594). Finalmente, gostaríamos de agradecer a Robert Clark, Dr. VenkateshKrishnan e Nathan Stadick para a sua avaliação crítica do manuscrito.

Este estudo foi realizado em estrita conformidade com as recomendações do Guia para o Cuidado e Uso de Animais de Laboratório do National Institutes of Health. O protocolo foi aprovado pela Universidade de Chicago Animal Care Institucional e Comitê de Uso (IACUC, números de protocolo 72066 e 72231). Todas as cirurgias foram realizadas sob anestesia, e todos os esforços foram feitos para minimizar o sofrimento. O células estaminais embrionárias humanas linha WA01 (H1; NIH-registro # 0043) foi adquirida da WiCell (Madison, WI) e cultivados utilizando o protocolo alimentador independente usando mTeSR1 mídia (Stem Cell Technologies, Vancouver, BC). Células-tronco embrionárias foram usadas dentro de dez passagens de descongelamento. 1. Isolamento do seio urogenital de rato ou o rato embriões <ol><li> Sacrificá um mouse cronometrado grávidas (17,5 dias) ou rato (18,5 dias) por inalação de CO 2 por 5 min. A morte é confirmada pelo desaparecimento dos sinais vitais do rato. Em seguida, quebrar o pescoço humanamente. O ra é sacrificado neste momento. Spray de etanol de 70% para esterilizar a pele abdominal e flancos. Cortar a pele abdominal e camadas musculares e, em seguida, cortou o útero, separar os embriões de ratos a partir do saco amniótico, e cortar o cordão umbilical. Transferir os embriões num tubo Falcon de 50 ml contendo solução salina equilibrada de Hank arrefecida com gelo (HBSS) e manter em gelo. Fetos são sacrificados por decapitação com uma tesoura cirúrgica. </li><li> Colocar o embrião 100 mm de Petri. Bissectar o embrião com uma tesoura, a nível do cordão umbilical (Figura 1A). Remover o estômago e intestino delgado. Revelam-se as ovários no embrião do sexo feminino e testículos no embrião do sexo masculino (Figura 1B e 1C). (Os ovários estão localizados nos pólos caudal dos rins. Os testículos são aproximadamente ao nível da bexiga). Neste estágio de desenvolvimento, UGSM isolado a partir ambos os embriões de machos e fêmeas são capazes de induzir uma linhagem epitelial prostática, na presença de acolhimentotestosterona. 4 Após este ponto o tempo de desenvolvimento, no entanto, os botões da próstata começam a se formar no UGSM, e isolamento de UGSM puro é muito desafiador. 4 </li><li> Sob o microscópio de dissecação, corte na parede abdominal na linha média com Vannas tesoura para expor a bexiga e parede posterior do abdome. Livre do trato genital superior de anexos (em embriões masculinos, corte o ureter, canal de Wolff e de Müller duto, em embriões do sexo feminino, corte o ureter e duto de Müller). Libertar a bexiga do cordão umbilical. Corte osso púbico e sem rodeios separada da parede anterior do seio urogenital com uma pinça de ponta fina Dumont. Finalmente, separar a parede posterior do seio urogenital, do recto. Corte do seio urogenital, com a bexiga na extremidade inferior (da uretra), e armazenar o seio urogenital em bloco (com a bexiga), em gelo HBSS frio. </li></ol> 2. A separação do seio urogenital Mesênquima <ol><li&gt; Remover a bexiga do seio urogenital sob o microscópio de dissecação (Figura 1D). </li><li> Transferência do seio urogenital para um tubo Falcon contendo 1% de tripsina em cálcio (Ca2 +) e magnésio (Mg 2 +) HBSS sem. Colocar o tubo no frigorífico a 4 ° C durante 75 min. </li><li> Remover a tripsina e neutralizar tripsinização com 10% de Soro Fetal Bovin (FBS) em meio DMEM/F12. </li><li> Cuidadosamente provocar a manga mesenquimal pegajoso do tubo epitelial com uma agulha ou Dumont pinça fina ponta (mantendo o tubo epitelial intacta reduz as hipóteses de contaminação das células epiteliais do mesênquima, o que pode resultar na formação de glândulas roedor, juntamente com a sua haste derivada de células humanas epitélio glandular) (Figura 1D) 12. </li><li> Transferir UGSM para um novo tubo Falcon contendo meio DMEM/F12 + 10% de FBS + 1% de Penicilina / Estreptomicina (Pen / Strep, 0,5 mg / ml) + 1% de não-essenciais Aminoácidos (NEAA), mais de 1 nM R1881. Place o tubo dentro de uma incubadora a 37 ° C com 5% de CO 2 durante a noite. </li><li> Centrifugar o tecido a 200 xg e desprezar o sobrenadante. Lava-se com tampão fosfato salino (PBS) (não perturbar o sedimento de tecido). </li><li> Adicionar 0,2% de colagenase (em DMEM/F12) para re-suspender o tecido. Incubar em 37 ° C com agitação suave durante 1 hora. </li><li> Vortex vigorosamente a digestão do tecido três vezes até que se torne mistura homogénea de uma única célula e adicionar meio DMEM/F12 + 10% de FBS + 1% de P / S 1% + NEAA R1881 1 nM + para neutralizar a colagenase. </li><li> Centrifugar a 200 xg e depois lave por re-suspender as células UGSM em HBSS, e repita três vezes para lavar completamente o UGSM. Finalmente suspender as células mesenquimais em DMEM/F12 e contar as células utilizando um hemocitómetro ou dispositivo de contagem de células. </li><li> Dissociar as células-tronco embrionárias humanas culta protocolo com alimentador-livre com mTeSR1 mídia (Stem Cell Technologies, Vancouver, BC), utilizando uma digestão Accutase (Millipore, Billerica, MA). Lave as células-tronco embrionárias humanasdurante a fase logarítmica do seu crescimento em meio DMEM/F12 quentes, em seguida, adicionar solução Accutase 1x quente, e incubar a 37 ° C durante 15-20 min, até que as células individuais são uniformemente dissociadas a partir de colónias. Adicionar uma quantidade igual de 1x definido inibidor de tripsina (Invitrogen, Grand Island, NY) para neutralizar o Accutase e pipeta suavemente para cima e para baixo para misturar e acabar com aglomerados de células. Células de pipeta para um tubo de centrífuga e centrifugar por 3 min a 200 xg, e re-suspender pellet celular em DMEM/F12 mídia. Centrifugar tubo por 3 min a 200 xg novamente e remover o sobrenadante, em seguida, re-suspensão em HBSS frio ou DMEM/F12 no volume desejado. </li><li> Adicionar células-tronco embrionárias humanas com relação de 1:2,5 hES / células mesenquimais. 3 Esta será uma composição celular de 1E 5 células UGSM com 4E 4 hESCs por 10 ml de injeção (um único rato UGS normalmente produz cerca de 2E 6 células mesenquimais). Centrifugar a 200 xg durante 5 min e, em seguida, eliminar o sobrenadante. Re-suspender, com 75% Matrigel (BD Biosciences,San Jose, CA, misturado com 25% HBSS frio para facilitar o manuseio) e coloque o tubo no gelo por injeção cápsula sub-renal. </li></ol> 3. Transplante de tecido recombinante Debaixo da cápsula renal <ol><li> Prepare a área cirúrgica esterilizada. </li><li> Anestesiar um rato nu atímica masculino com ip cetamina / xilazina; usar um mix fresco contendo uma proporção de 01:01:08 ketamina: xilazina: soro fisiológico. A dosagem será de 100 mg / kg de cetamina e 2,5 mg / kg de xilazina. Animais que não respondem a um dedo do pé-pitada deve ser totalmente sob por 20-30 min. </li><li> Cirurgicamente preparar o mouse raspando o local da cirurgia (se não estiver usando um mouse nu), limpeza com três panos alternados de álcool a 70%, seguido por solução betadine e aplicação de um campo cirúrgico. </li><li> Coloque olho umidade pomada (Altalube pomada) para os olhos do mouse para evitar o ressecamento durante a anestesia. </li><li> Durante a cirurgia, a taxa de respiração e temperatura corporal dos ratinhos foram monitorizados. As taxas de respiração shoULD ser estável e mantida dentro de 40-90 respirações / min. A temperatura corporal central é monitorizado com observação da cor das membranas mucosas e as solas dos pés-de-rosa. </li><li> Faça uma incisão na pele cm de comprimento 1,0 longitudinal na parte de trás. Continuar a cortar a parede abdominal, numa incisão longitudinal de 0,5 cm. </li><li> Aperte suavemente o rim para fora do abdômen e manter a umidade da superfície renal usando gotas de solução salina estéril. </li><li> Gentilmente perfurar a cápsula renal usando uma agulha de seringa de calibre 27 vazios. A punção muito superficial é suficiente. Este será o lugar onde você inserir a agulha sem corte para injetar sua mistura de celular. </li><li> Encha uma agulha de seringa sem corte de calibre 28, com 10 ml de sua mistura de células. Lentamente inserir o local de punção e penetrar no parênquima renal para alcançar uma área por baixo da cápsula renal. Injectam-se 10 ul da mistura celular para formar um bolo no rim sob a cápsula renal. Retirada da agulha. </li><li> Retorne o rim à abdocavidade minal. Fixe a camada muscular do peritônio com fio de náilon 4-0. </li><li> Feche a pele ou com sutura ou grampo. </li><li> Limpar o sangue a partir da pele utilizando solução salina estéril. </li></ol> 4. Analgesia pós-operatória e Monitoramento <ol><li> Injectar o animal com buprenorfina (0,1 mg / kg a cada 12 horas) ou cetoprofeno (3-5 mg / kg em salina a cada 12 horas), para controlar a dor pós-operatória, e 1 ml de 37 ° C, solução salina IP para manter o animal aquecido e evitar a desidratação. </li><li> Coloque o animal em uma gaiola limpa e coloque-o sobre uma almofada de aquecimento suave. </li><li> Envie os animais de volta para a sala de animais, uma vez que recuperar a consciência e estão se movendo dentro da gaiola. </li><li> Observar o animal diariamente para sinais inesperados de doença e de infecção para os três dias seguintes. Os ratos são monitorados com a observação de atividades em gaiolas, incluindo comida e água acessando um dia de pós-operatório. Se os ratos exibir menos atividades, cetoprofeno é intraperitoneal anúncioministrou novamente uma vez por dia. </li><li> Remova os grampos de pele ou suturas duas semanas após a cirurgia. </li><li> Xenoenxerto tecidos podem ser colhidas mais cedo 8 semanas pós-cirurgia. Os xenoenxertos pode ser trabalhada in vivo utilizando ultra-sons de pequenos animais (Figura 2A), e os tecidos fixos pode ser seccionada e corada para proteínas utilizando vários imuno-histoquímica (Figura 3). </li></ol>

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Os autores não têm qualquer conflito de interesse com o trabalho apresentado.

Recombinação de tecido, utilizando UGSM é uma técnica extremamente útil para investigar o desenvolvimento da próstata e os acontecimentos moleculares que conduzem a cancro da próstata iniciação. O potencial indutor de UGSM foi usado para numerosas aplicações na investigação da próstata, que incluem a melhoria do tumor ter de linhas de células da próstata e tumores, estudar interacções estromal-epiteliais, e formando próstata recombinantes inter-espécies 7,17-20 preparação adequada de UGS...

Há uma tremenda necessidade de melhores sistemas de modelos humanas de câncer de próstata. Em particular, os sistemas modelo humanos relevantes de tecidos normais da próstata, não malignas que podem ser geneticamente manipulados para discernir directamente o papel de genes específicos no início do cancro da próstata seria extremamente informativo. O advento da era genômica identificou vários genes que podem ter um papel na formação do câncer. A falta de sistemas de modelos experimentais humanos, no entanto, prejudica gravemente a nossa capacidade de testar e caracterizar funcionalmente candidatos genes de susceptibilidade ao câncer de próstata. Um sistema modelo ideal facilitaria as análises funcionais rápidas e mais rápida de genes de susceptibilidade de cancro em combinação com os sistemas modelo de roedores transgénicos adequados. Além disso, tal sistema modelo humano permitiria a caracterização molecular dos mecanismos de sinalização de carcinogênese da próstata em direção à descoberta e validação de novas terapias paraevitar a formação de cancro da próstata. Células-tronco embrionárias humanas (hESCs) são capazes de formar tecidos humanos prostáticas como xenografts. Em 2006, Taylor, et ai. HESCs relatado que podem ser induzidas a formar epitélio da próstata in vivo, quando re-combinado com o roedor mesênquima do seio urogenital (UGSM) dentro de um período de 8-12 semanas. Uma Estes estudos foram baseados em trabalhos anteriores por o laboratório Cunha mostrando que roedor UGSM embrionário pode promover a diferenciação de células da próstata e as populações de células estaminais embrionárias epiteliais in vivo. 2,3 A próstata se desenvolve a partir de um denominado Anlagen embrionária do seio urogenital (UGS), e antes do dia embrionário 17 (E17 rato ; E18 dia no rato) os UGS pode ser fisicamente removido e dividido em epitélio (UGSE) e mesênquima (UGSM) 4 Essa abordagem recombinação tecido aumentou significativamente a nossa compreensão do desenvolvimento e carcinogénese da próstata, em particular.factor de crescimento ly e vias de sinalização hormonais e as relações moleculares entre epitélio e estroma da próstata. 5-8 Este método envolve a combinação de UGSM ex vivo, com as células estaminais epiteliais ou da mesma ou de espécies distintas e estes recombinantes celulares / tecido e são implantados cultivados e xenoenxertos de dentro do rato hospedeiro. 4,9 Após um período de crescimento in vivo, o implante contém estruturas epiteliais glandulares da próstata incorporados no tecido estromal. Mais de coloração pode ser realizado para determinar se tais estruturas são verdadeiramente prostática e de origem humana 10,11.

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Hank's Balanced Salt Solution (HBSS)</td>
 <td>GIBCO</td>
 <td>14170</td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM/F12</td>
 <td>GIBCO</td>
 <td>11330</td>
 <td></td>
 </tr>
 <tr>
 <td>R1881</td>
 <td>Sigma</td>
 <td>965-93-5</td>
 <td>Mix to 1 ug/ml in Ethanol (1,000x stock)</td>
 </tr>
 <tr>
 <td>NEAA</td>
 <td>GIBCO</td>
 <td>11140</td>
 <td></td>
 </tr>
 <tr>
 <td>Pen-Strep Solution</td>
 <td>GIBCO</td>
 <td>15070</td>
 <td>100x stock</td>
 </tr>
 <tr>
 <td>Matrigel</td>
 <td>BD Biosciences</td>
 <td>354230</td>
 <td></td>
 </tr>
 <tr>
 <td>KETASET (ketamine hydrochloride) </td>
 <td>Fort Dodge Animal Health</td>
 <td>NDC 0856-2013-01</td>
 <td>100 mg/ml; dilute 1:10 in sterile saline </td>
 </tr>
 <tr>
 <td>AnaSed (xylazine) </td>
 <td>VET-A-MIX, Inc.</td>
 <td>NADA 139-236</td>
 <td>20 mg/ml; dilute 1:10 in sterile saline</td>
 </tr>
 <tr>
 <td>Trypsin</td>
 <td>BD Biosciences</td>
 <td>215240</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase </td>
 <td>Sigma</td>
 <td>C2014</td>
 <td></td>
 </tr>
 <tr>
 <td>Ketoprofen </td>
 <td>Fort Dodge</td>
 <td>NDC 0856-4396-01</td>
 <td>100 mg/ml; dilute 1:1,000 in sterile saline</td>
 </tr>
 <tr>
 <td>Altalube eye ointment </td>
 <td>Altaire Pharmaceuticals, Inc.</td>
 <td>NLC 56641-19850</td>
 <td></td>
 </tr>
 <tr>
 <td>Leica MZ16 F Stereomicroscope</td>
 <td>Leica</td>
 <td></td>
 <td>Any good dissecting scope can be used.</td>
 </tr>
 <tr>
 <td>Vannas spring scissors</td>
 <td>Fine Science Tools</td>
 <td>15001-08</td>
 <td></td>
 </tr>
 <tr>
 <td>Syringe</td>
 <td>Hamilton</td>
 <td>84855</td>
 <td></td>
 </tr>
 <tr>
 <td>Hamilton Needle, Small RN, 28 gauge, 0.5inches, Point Style #3 (Blunt)</td>
 <td>Hamilton</td>
 <td>7803-02 </td>
 <td>Custom Needle </td>
 </tr>
 <tr>
 <td>Ethanol Prep Pads</td>
 <td>Fisher Scientific</td>
 <td>06-669-62 </td>
 <td></td>
 </tr>
 <tr>
 <td>Sterile Gauze Pads</td>
 <td>Fisher Scientific</td>
 <td>22-415-469 </td>
 <td></td>
 </tr>
 <tr>
 <td>Ethicon Vicryl Suture (4-0 FS-2)</td>
 <td>MedVet International</td>
 <td>J392H </td>
 <td>Needle-in, dissolvable suture </td>
 </tr>
 <tr>
 <td>Autoclip 9 mm Wound Clips</td>
 <td>Becton Dickenson</td>
 <td>427631</td>
 <td></td>
 </tr>
 <tr>
 <td>PVP Iodine Prep Pads</td>
 <td>Fisher Scientific</td>
 <td>06-669-98 </td>
 <td></td>
 </tr>
 <tr>
 <td>Dissector scissor with blunt end</td>
 <td>Fine Science Tools</td>
 <td>14072-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Dumont fine tip forceps</td>
 <td>Fine Science Tools</td>
 <td>11252-50</td>
 <td></td>
 </tr>
 <tr>
 <td>Needle holder with Scissor</td>
 <td>Fine Science Tools</td>
 <td>12002-14</td>
 <td></td>
 </tr>
 </tbody>
</table>

formation of human prostate epithelium using tissue recombination of rodent urogenital sinus mesenchyme and human stem cells

Avanços na pesquisa sobre o câncer de próstata é severamente limitada pela disponibilidade de sistemas-modelo de origem humana e hormônio-naïve, que limitam a nossa capacidade de compreender os eventos genéticos e moleculares subjacentes próstata iniciação doença. Para o desenvolvimento de melhores sistemas de modelo para o estudo da próstata humana carcinogênese, nós e outros aproveitaram o único potencial indutivo pró-prostática de embriões de roedores próstata estroma, denominado mesênquima do seio urogenital (UGSM). Quando recombinado com certas populações de células, tais como células pluripotentes estaminais embrionárias, UGSM induz a formação de epitélio da próstata humana normal de uma forma dependente da testosterona. Tal sistema modelo humano pode ser utilizado para investigar experimentalmente e testar a capacidade dos candidatos genes de susceptibilidade de cancro da próstata a um ritmo acelerado em comparação com os estudos de roedores transgénicos típicos. Uma vez que as células-tronco embrionárias humanas (hESCs) pode ser geneticamente modificado em cultura using de expressão do gene indutivel ou siRNA knock-down vectores anteriores à recombinação dos tecidos, tal modelo facilita os testes das consequências funcionais de genes ou combinações de genes, que são pensados ​​para promover ou prevenir a carcinogénese. A técnica de isolamento de populações puras de células UGSM, no entanto, é um desafio e aprendizagem muitas vezes requer a alguém com experiência anterior ensinar pessoalmente. Além disso, a inoculação de misturas de células sob a cápsula renal de um hospedeiro imunocomprometido pode ser tecnicamente desafiadora. Aqui destacamos e ilustrar adequado isolamento de UGSM a partir de embriões de roedores e implantação cápsula renal de misturas de tecidos para formar epitélio da próstata humana. Tal abordagem, em seu estágio atual, requer in vivo xeno de células-tronco embrionárias; aplicações futuras poderiam incluir na formação da glândula vitro ou a utilização de populações de células-tronco pluripotentes induzidas (iPSCs).

Baseando-se no relatório emocionante por Taylor, et al., Nosso laboratório desenvolveu um protocolo de enxerto usando o H1 utilizado (NIH-designado WA01, geneticamente masculino) linha de células estaminais embrionárias humanas. 1 Esta linha tem sido rigorosamente testados para o controle de qualidade e é normal, karyotypically 13 quando cultivadas de forma adequada, as células-tronco embrionárias humanas pode ser mantida, ampliada e criopreservados em um estado indiferenciado e plu...

Watch this Scientific Journal Video about Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells at JoVE.com

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Para desvendar são desesperadamente necessários os primeiros mecanismos moleculares subjacentes câncer de próstata iniciação, romance e sistemas de modelos humanos e abordagens inovadoras. O potencial de pré-prostática seio urogenital mesênquima (UGSM) para induzir as populações de células estaminais pluripotentes para formar o epitélio da próstata humana é uma ferramenta poderosa na investigação experimental próstata.

Formação de Epithelium próstata humano utilizando Tissue Recombinação de Rodent Urogenital Mesênquima Sinus e células-tronco humanas

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

formation-human-prostate-epithelium-using-tissue-recombination-rodent

Research

JoVE Journal

Medicine

13.6K visualizações.  University of Chicago. Para desvendar são desesperadamente necessários os primeiros mecanismos moleculares subjacentes câncer de próstata iniciação, romance e sistemas de modelos humanos e abordagens inovadoras. O potencial de pré-prostática seio urogenital mesênquima (UGSM) para induzir as populações de células estaminais pluripotentes para formar o epitélio da próstata humana é uma ferramenta poderosa na investigação experimental próstata.

Vídeo: Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

Transplante de Células-tronco em um In vitro Modelo de isquemia / reperfusão Simulated

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Medicina

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

Injeção de sub-retiniana de Vetores da terapia genética e células-tronco no olho do rato

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Isolamento, caracterização e comparativa Diferenciação de células-tronco da polpa dentária Derivado de dentes permanentes usando dois métodos diferentes

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Um Modelo murino de próstata humana Metástase Ortotópico

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

Isolamento de células-tronco cancerosas em amostras de câncer de próstata humano

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

Avaliação das Propriedades Stem Cell em carcinomas de células do ovário humano utilizar multi e individuais baseados em células Esferas Ensaios

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types. Healthy prostate is comprised of fibromuscular stroma surrounding discrete epithelial-lined secretory lumens and a very small population of immune and neuroendocrine cells. In contrast, areas of prostate cancer have increased dysplastic luminal epithelium with greatly reduced or absent stromal population. Given the profound difference between stromal and epithelial cell types, it is imperative to separate the cell types for any type of downstream molecular analysis. Despite this knowledge, the bulk of gene expression studies compare benign prostate to cancer without micro-dissection, leading to stromal bias in the benign samples. Laser-capture micro-dissection (LCM) is an effective method to physically separate different cell types from a specimen section. The goal of this protocol is to show that RNA can be successfully isolated from LCM-collected human prostatic epithelium and used for downstream gene expression studies such as RT-qPCR and RNAseq.

The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

Captura de Laser Microdissection de Próstata Humano Epithelium para Análise de RNA

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types.  Healthy prostate is comprised of ...

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

Pré-clínica Ortotópico Modelo murino de cancro da próstata humano

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in which the liver has been repopulated with functional human hepatocytes, could serve as a useful tool for investigating human hepatic cell biology, drug metabolism, and other preclinical applications. One of the key factors required for successful transplantation of human hepatocytes into mice is the elimination of the endogenous hepatocytes to prevent competition with the human cells and provide a suitable space and microenvironment for promoting human donor cell expansion and differentiation. To date, two major liver injury mouse models utilizing fumarylacetoacetate hydrolase (Fah) and uroplasminogen activator (uPA) mice have been established. However, Fah mice are used mainly with mature hepatocytes and the application of the uPA model is limited by decreased breeding. To overcome these limitations, Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice were used for in vivo differentiation of immature human hepatocytes and humanized liver generation. Human hepatic stem cells (HpSCs) successfully repopulated the livers of Alb-TRECK/SCID mice that had developed lethal fulminant hepatic failure following diphtheria toxin (DT) treatment. This model of a humanized liver in Alb-TRECK/SCID mice will have functional applications in studies involving drug metabolism and drug-drug interactions and will promote other in vivo and in vitro studies.

Here, we present a novel humanized mouse liver model generated in Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice following the transplantation of immature and expandable human hepatic stem cells.

Geração de um humanizado rato Liver Usando células-tronco hepáticas humanas

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

Avaliação da função Microvascular de tecido adiposo humano usando tele-microscopia

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...