RESUMEN
To evaluate hematopoietic niche cell populations isolated from human embryonic stem cells (hESCs), we tested the ability of hESC-derived stromal lines to support CD34(+) umbilical cord blood (UCB)- and hESC-derived CD34(+)45(+) cells in long-term culture initiating cell (LTC-IC) assays. Specifically, these hematopoietic populations were cocultured with hESC-derived mesenchymal stromal cells (hESC-MSCs) and hESC-derived endothelial cells (hESC-ECs), and then assessed for their LTC-IC potential in comparison to coculture with bone marrow (BM)-derived MSCs and the mouse stromal line M2-10B4. We found that the hESC-derived stromal lines supported LTC-ICs from UCB similar to M2-10B4 cells and better than BM-MSCs. However, none of the stromal populations supported LTC-IC from hESC-derived CD34(+)45(+) cells. Engraftment data using the output from LTC-IC assays showed long-term repopulation (12 weeks) of NSG mice to correlate with LTC-IC support on a given stromal layer. Therefore, hESC-derived stromal lines can be used to efficiently evaluate putative hematopoietic stem/progenitor cells derived from hESCs or other cell sources.
Asunto(s)
Técnicas de Cultivo de Célula , Células Madre Embrionarias/citología , Sangre Fetal/citología , Células Madre Hematopoyéticas/citología , Animales , Antígenos CD34/metabolismo , Células de la Médula Ósea/citología , Linaje de la Célula , Técnicas de Cocultivo , Ensayo de Unidades Formadoras de Colonias , Células Madre Embrionarias/metabolismo , Sangre Fetal/metabolismo , Células Madre Hematopoyéticas/metabolismo , Humanos , Antígenos Comunes de Leucocito/metabolismo , Células Madre Mesenquimatosas/citología , Ratones , Células del Estroma/citologíaRESUMEN
Adoptive transfer of antitumor lymphocytes has gained intense interest in the field of cancer therapeutics over the past two decades. Human natural killer (NK) cells are a promising source of lymphocytes for anticancer immunotherapy. NK cells are part of the innate immune system and exhibit potent antitumor activity without need for human leukocyte antigen matching and without prior antigen exposure. Moreover, the derivation of NK cells from pluripotent stem cells could provide an unlimited source of lymphocytes for off-the-shelf therapy. To date, most studies on hematopoietic cell development from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have used incompletely defined conditions and been on a limited scale. Here, we have used a two-stage culture system to efficiently produce NK cells from hESCs and iPSCs in the absence of cell sorting and without need for xenogeneic stromal cells. This novel combination of embryoid body formation using defined conditions and membrane-bound interleukin 21-expressing artificial antigen-presenting cells allows production of mature and functional NK cells from several different hESC and iPSC lines. Although different hESC and iPSC lines had varying efficiencies in hematopoietic development, all cell lines tested could produce functional NK cells. These methods can be used to generate enough cytotoxic NK cells to treat a single patient from fewer than 250,000 input hESCs/iPSCs. Additionally, this strategy provides a genetically amenable platform to study normal NK cell development and education in vitro.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Células Madre Pluripotentes Inducidas/citología , Células Asesinas Naturales/citología , Neoplasias/terapia , Animales , Células Presentadoras de Antígenos/citología , Línea Celular , Proliferación Celular , Cuerpos Embrioides/citología , Células Nutrientes/citología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Inmunoterapia , Ratones , Neoplasias/inmunología , Células del Estroma/citologíaRESUMEN
Efficient derivation and isolation of hematopoietic stem cells (HSCs) from human pluripotent stem cell (hPSC) populations remains a major goal in the field of developmental hematopoiesis. These enticing pluripotent stem cells (comprising both human embryonic stem cells and induced pluripotent stem cells) have been successfully used to generate a wide array of hematopoietic cells in vitro, from primitive hematoendothelial precursors to mature myeloid, erythroid, and lymphoid lineage cells. However, to date, PSC-derived cells have demonstrated only limited potential for long-term multilineage hematopoietic engraftment in vivo - the test by which putative HSCs are defined. Successful generation and characterization of HSCs from hPSCs not only requires an efficient in vitro differentiation system that provides insight into the developmental fate of hPSC-derived cells, but also necessitates an in vivo engraftment model that allows identification of specific mechanisms that hinder or promote hematopoietic engraftment. In this chapter, we will describe a method that utilizes firefly luciferase-expressing hPSCs and bioluminescent imaging to noninvasively track the survival, proliferation, and migration of transplanted hPSC-derived cells. Combined with lineage and functional analyses of engrafted cells, this system is a useful tool to gain insight into the in vivo potential of hematopoietic cells generated from hPSCs.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Células Madre Hematopoyéticas/citología , Células Madre Pluripotentes/citología , Animales , Células Sanguíneas/citología , Células de la Médula Ósea/citología , Diferenciación Celular , Separación Celular , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Humanos , Imagenología Tridimensional , Hígado/citología , Hígado/metabolismo , Luciferasas/metabolismo , Mediciones Luminiscentes , Ratones , Células Madre Pluripotentes/metabolismo , Bazo/citologíaRESUMEN
A novel anticancer agent was constructed by fusing a gene encoding the scFV that targets both glycosylated and unglycosylated forms of CD133 to a gene fragment encoding deimmunized PE38KDEL. The resulting fusion protein, dCD133KDEL, was studied to determine its ability to bind and kill tumor-initiating cells in vitro and in vivo. The anti-CD133 scFV selectively bound HEK293 cells transfected with the CD133 receptor gene. Time course viability studies showed that dCD133KDEL selectively inhibited NA-SCC and UMSCC-11B, 2 head and neck squamous cell carcinomas that contain a CD133 expressing subpopulation. Importantly, the drug did not inhibit the viability of hematopoietic lineages measured by long-term culture-initiating cell and colony-forming assays from sorted human CD34+ progenitor cells. In addition to in vitro studies, in vivo tumor initiation experiments confirmed that CD133-sorted cells implanted into the flanks of nude mice grew faster and larger than unsorted cells. In contrast, cells that were pretreated with dCD133KDEL before implantation showed the slowest and lowest incidence of tumors. Furthermore, UMSCC-11B-luc tumors treated with multiple intratumoral injections of dCD133KDEL showed marked growth inhibition, leading to complete degradation of the tumors that was not observed with an irrelevant control-targeted toxin. Experiments in immunocompetent mice showed that toxin deimmunization resulted in a 90% reduction in circulating antitoxin levels. These studies show that dCD133KDEL is a novel anticancer agent effective at inhibiting cell proliferation, tumor initiation, and eliminating established tumors by targeting the CD133 subpopulation. This agent shows significant promise for potential development as a clinically useful therapy.
Asunto(s)
Antígenos CD/inmunología , Carcinoma de Células Escamosas/tratamiento farmacológico , Glicoproteínas/inmunología , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/patología , Inmunotoxinas/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Péptidos/inmunología , Proteínas Recombinantes de Fusión/farmacología , Antígeno AC133 , ADP Ribosa Transferasas/genética , ADP Ribosa Transferasas/farmacocinética , ADP Ribosa Transferasas/farmacología , Animales , Antígenos CD/biosíntesis , Antígenos CD/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/farmacocinética , Toxinas Bacterianas/farmacología , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Exotoxinas/genética , Exotoxinas/farmacocinética , Exotoxinas/farmacología , Femenino , Glicoproteínas/biosíntesis , Glicoproteínas/genética , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/farmacología , Inmunotoxinas/genética , Inmunotoxinas/farmacocinética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Péptidos/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacocinética , Transfección , Factores de Virulencia/genética , Factores de Virulencia/farmacocinética , Factores de Virulencia/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Exotoxina A de Pseudomonas aeruginosaRESUMEN
Cell-based therapies against HIV/AIDS have been gaining increased interest. Natural killer (NK) cells are a key component of the innate immune system with the ability to kill diverse tumor cells and virus-infected cells. While NK cells have been shown to play an important role in the control of HIV-1 replication, their functional activities are often compromised in HIV-1-infected individuals. We have previously demonstrated the derivation of NK cells from human embryonic stem cells (hESCs) with the ability to potently kill multiple types of tumor cells both in vitro and in vivo. We now demonstrate the derivation of functional NK cells from human induced pluripotent stem cells (iPSCs). More importantly, both hESC- and iPSC-derived NK cells are able to inhibit HIV-1 NL4-3 infection of CEM-GFP cells. Additional studies using HIV-1-infected human primary CD4(+) T cells illustrated that hESC- and iPSC-derived NK cells suppress HIV-1 infection by at least three distinct cellular mechanisms: killing of infected targets through direct lysis, antibody-dependent cellular cytotoxicity, and production of chemokines and cytokines. Our results establish the potential to utilize hESC- and iPSC-derived NK cells to better understand anti-HIV-1 immunity and provide a novel cellular immunotherapeutic approach to treat HIV/AIDS.
Asunto(s)
Citotoxicidad Inmunológica , Infecciones por VIH/inmunología , VIH-1/inmunología , Células Madre Pluripotentes Inducidas/citología , Células Asesinas Naturales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Diferenciación Celular , Línea Celular , Células Cultivadas , Células Madre Embrionarias/citología , Células Madre Embrionarias/inmunología , Infecciones por VIH/virología , VIH-1/fisiología , Humanos , Células Madre Pluripotentes Inducidas/inmunología , Células Madre Pluripotentes Inducidas/fisiología , Células Asesinas Naturales/citología , Activación de Linfocitos , Linfocitos T , Replicación Viral/inmunologíaRESUMEN
Human embryonic stem cells (hESCs) provide an important resource for novel regenerative medicine therapies and have been used to derive diverse cell populations, including hematopoietic and endothelial cells. However, it remains a challenge to achieve significant engraftment of hESC-derived blood cells when transplanted into animal models. To better understand mechanisms that enhance or limit the in vivo developmental potential of hESC-derived cells, we utilized hESCs that express firefly luciferase (luc) to allow noninvasive, real-time bioluminescent imaging of hESC-derived CD34(+) cells transplanted into the liver of neonatal immunodeficient mice. Serial imaging demonstrated stable engraftment and expansion of the luc(+) hESC-derived cells in vivo over several months. While we found that these hESC-derived CD34(+) cells have bipotential ability to generate both hematopoietic and endothelial lineages in vitro, these studies demonstrate preferential differentiation into endothelial cells in vivo, with only low levels of hematopoietic cell engraftment. Therefore, these studies reveal key differences in the developmental potential of hESC-derived cells using in vitro and in vivo analyses. Although transplanted hESC-derived CD34(+) cells are well-suited for revascularization therapies, additional measures are needed to provide higher levels of long-term hematopoietic engraftment.