Xinghui Tian

Hematopoietic Engraftment of Human Embryonic Stem Cell- Derived Cells Is Regulated by Recipient Innate Immunity

Human embryonic stem cells (hESCs) provide an important means to characterize early stages of hematopoietic development. However, the in vivo potential of hESC‐derived hematopoietic cells has not been well defined. We demonstrate that hESC‐derived cells are capable of long‐term hematopoietic engraftment when transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Human CD45+ and CD34+ cells are identified in the mouse bone marrow (BM) more than 3 months after injection of hESCs that were allowed to differentiate on S17 stromal cells for 7–24 days. Secondary engraftment studies further confirm long‐term repopulating cells derived from hESCs. We also evaluated two mechanisms that may inhibit engraftment: host immunity and requirement for homing to BM. Treatment with anti‐ASGM1 antiserum that primarily acts by depletion of natural killer cells in transplanted mice leads to improved engraftment, likely due to low levels of HLA class I expressed on hESCs and CD34+ cells derived from hESCs. Intra‐BM injection also provided stable engraftment, with hematopoietic cells identified in both the injected and contra‐lateral femur. Importantly, no teratomas are evident in animals injected with differentiated hESCs. These results demonstrate that SCID‐repopulating cells, a close surrogate for hematopoietic stem cells, can be derived from hESCs. Moreover, both adaptive and innate immune effector cells may be barriers to engraftment of these cells.

Human embryonic stem cells differentiate into a homogeneous population of natural killer cells with potent in vivo antitumor activity

Natural killer (NK) cells serve as important effectors for antitumor immunity, and CD56+CD45+ NK cells can be routinely derived from human embryonic stem cells (hESCs). However, little is know about the ability of hESC-derived NK cells to mediate an effective in vivo antitumor response. Using bioluminescent imaging, we now demonstrate that H9 line hESC-derived NK cells mediate effective clearance of human tumor cells in vivo. In addition to increased in vitro killing of diverse tumor targets, the in vivo tumor clearance by H9 hESC-derived NK cells was more effective compared with NK cells derived from umbilical cord blood (UCB). Phenotypic analysis demonstrates the hESC-derived NK cells are uniformly CD94+CD117(low/-), an NK-cell population characterized by potent cytolytic activity and thus more competent to mediate tumor clearance. These studies demonstrate that hESCs provide an important model to study human lymphocyte development and may serve as a novel source for antitumor immunotherapy.

Efficient and Stable Transgene Expression in Human Embryonic Stem Cells Using Transposon‐Mediated Gene Transfer

Efficient and stable genetic modification of human embryonic stem (ES) cells is required to realize the full scientific and potential therapeutic use of these cells. Currently, only limited success toward this goal has been achieved without using a viral vector. The Sleeping Beauty (SB) transposon system mediates nonviral gene insertion and stable expression in target cells and tissues. Here, we demonstrate use of the nonviral SB transposon system to effectively mediate stable gene transfer in human ES cells. Transposons encoding (a) green fluorescent protein coupled to the zeocin gene or (b) the firefly luciferase (luc) gene were effectively delivered to undifferentiated human ES cells with either a DNA or RNA source of transposase. Only human ES cells cotransfected with transposon‐ and transposase‐encoding sequences exhibited transgene expression after 1 week in culture. Molecular analysis of transposon integrants indicated that 98% of stable gene transfer resulted from transposition. Stable luc expression was observed up to 5 months in human ES cells cotransfected with a transposon along with either DNA or RNA encoding SB transposase. Genetically engineered human ES cells demonstrated the ability to differentiate into teratomas in vivo and mature hematopoietic cells in vitro while maintaining stable transgene expression. We conclude that the SB transposon system provides an effective approach with several advantages for genetic manipulation and durable gene expression in human ES cells.

Hematopoietic Development of Human Embryonic Stem Cells in Culture

The successful isolation and characterization of human embryonic stem cells (hESCs) provides a powerful tool to study the cellular and genetic mechanisms that mediate cell-fate decisions toward distinct developmental lineages. hESC-derived cells may also be suitable for novel cellular therapies. Significant progress in hematopoietic development of hESCs has demonstrated production of many types of blood cells from hESCs including myeloid, erythroid and lymphoid lineage cells, and possibly hematopoietic stem cells. Current established approaches to generate specific hematopoietic lineages are based on the initial pre-differentiation of hESCs into a heterogeneous mixture of cell populations. In this chapter, we describe two methods that have been successfully used in our laboratory: (1) co-culture with stromal cells derived from hematopoietic microenvironments and (2) x embryoid body (EB) formation. Subsequent to this early differentiation step, distinct progenitor cell populations can be derived, sorted, and utilized for further lineage-specific developmental studies.

Bioluminescent Imaging Demonstrates That Transplanted Human Embryonic Stem Cell-Derived CD34+ Cells Preferentially Develop into Endothelial Cells†‡§

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. STEM CELLS 2009;27:2675–2685

Differentiation of embryonic stem cells towards hematopoietic cells: progress and pitfalls.

Purpose of reviewHematopoietic development from embryonic stem cells has been one of the most productive areas of stem cell biology. Recent studies have progressed from work with mouse to human embryonic stem cells. Strategies to produce defined blood cell populations can be used to better understand normal and abnormal hematopoiesis, as well as potentially improve the generation of hematopoietic cells with therapeutic potential. Recent findingsMolecular profiling, phenotypic and functional analyses have all been utilized to demonstrate that hematopoietic cells derived from embryonic stem cells most closely represent a stage of hematopoiesis that occurs at embryonic/fetal developmental stages. Generation of hematopoietic stem/progenitor cells comparable to hematopoietic stem cells found in the adult sources, such as bone marrow and cord blood, still remains challenging. However, genetic manipulation of intrinsic factors during hematopoietic differentiation has proven a suitable approach to induce adult definitive hematopoiesis from embryonic stem cells. SummaryConcrete evidence has shown that embryonic stem cells provide a powerful approach to study the early stage of hematopoiesis. Multiple hematopoietic lineages can be generated from embryonic stem cells, although most of the evidence suggests that hematopoietic development from embryonic stem cells mimics an embryonic/fetal stage of hematopoiesis.

In Vivo Evaluation of Putative Hematopoietic Stem Cells Derived from Human Pluripotent Stem Cells

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.

In vivo selection of human embryonic stem cell-derived cells expressing methotrexate-resistant dihydrofolate reductase

Human embryonic stem cells (hESCs) provide a novel source of hematopoietic and other cell populations suitable for gene therapy applications. Preclinical studies to evaluate engraftment of hESC-derived hematopoietic cells transplanted into immunodeficient mice demonstrate only limited repopulation. Expression of a drug-resistance gene, such as Tyr22-dihydrofolate reductase (Tyr22-DHFR), coupled to methotrexate (MTX) chemotherapy has the potential to selectively increase the engraftment of gene-modified, hESC-derived cells in mouse xenografts. Here, we describe the generation of Tyr22-DHFR–GFP-expressing hESCs that maintain pluripotency, produce teratomas and can differentiate into MTXr-hemato-endothelial cells. We demonstrate that MTX administered to nonobese diabetic/severe combined immunodeficient/IL-2Rγcnull (NSG) mice after injection of Tyr22-DHFR-hESC-derived cells significantly increases human CD34+ and CD45+ cell engraftment in the bone marrow (BM) and peripheral blood of transplanted MTX-treated mice. These results demonstrate that MTX treatment supports selective, long-term engraftment of Tyr22-DHFR cells in vivo, and provides a novel approach for combined human cell and gene therapy.