Key Points• Expression of RUNX1a, an isoform of RUNX1, enhances blood cell production from human pluripotent stem cells.Advancements in human pluripotent stem cell (hPSC) research have potential to revolutionize therapeutic transplantation. It has been demonstrated that transcription factors may play key roles in regulating maintenance, expansion, and differentiation of hPSCs. In addition to its regulatory functions in hematopoiesis and blood-related disorders, the transcription factor RUNX1 is also required for the formation of definitive blood stem cells. In this study, we demonstrated that expression of endogenous RUNX1a, an isoform of RUNX1, parallels with lineage commitment and hematopoietic emergence from hPSCs, including both human embryonic stem cells and inducible pluripotent stem cells. In a defined hematopoietic differentiation system, ectopic expression of RUNX1a facilitates emergence of hematopoietic progenitor cells (HPCs) and positively regulates expression of mesoderm and hematopoietic differentiation-related factors, including Brachyury, KDR, SCL, GATA2, and PU.1. HPCs derived from RUNX1a hPSCs show enhanced expansion ability, and the ex vivo-expanded cells are capable of differentiating into multiple lineages. Expression of RUNX1a in embryoid bodies (EBs) promotes definitive hematopoiesis that generates erythrocytes with b-globin production. Moreover, HPCs generated from RUNX1a EBs possess ‡9-week repopulation ability and show multilineage hematopoietic reconstitution in vivo. Together, our results suggest that RUNX1a facilitates the process of producing therapeutic HPCs from hPSCs. (Blood. 2013;121(15):2882-2890

The sea urchin larval skeleton offers a simple model for formation of developmental patterns. The calcium carbonate skeleton is secreted by primary mesenchyme cells (PMCs) in response to largely unknown patterning cues expressed by the ectoderm. To discover novel ectodermal cues, we performed an unbiased RNA-Seq-based screen and functionally tested candidates; we thereby identified several novel skeletal patterning cues. Among these, we show that SLC26a2/7 is a ventrally expressed sulfate transporter that promotes a ventral accumulation of sulfated proteoglycans, which is required for ventral PMC positioning and skeletal patterning. We show that the effects of SLC perturbation are mimicked by manipulation of either external sulfate levels or proteoglycan sulfation. These results identify novel skeletal patterning genes and demonstrate that ventral proteoglycan sulfation serves as a positional cue for sea urchin skeletal patterning.

Derivation of hematopoietic stem cells from human pluripotent stem cells remains a key goal for the fields of developmental biology and regenerative medicine. Here, we use a novel genetic reporter system to prospectively identify and isolate early hematopoietic cells derived from human embryonic stem cells (hESCs) and human induced pluripotent cells (iPSCs). Cloning the human RUNX1c P1 promoter and +24 enhancer to drive expression of tdTomato (tdTom) in hESCs and iPSCs, we demonstrate that tdTom expression faithfully enriches for RUNX1c-expressing hematopoietic progenitor cells. Time-lapse microscopy demonstrated the tdTom+ hematopoietic cells to emerge from adherent cells. Furthermore, inhibition of primitive hematopoiesis by blocking Activin/Nodal signaling promoted the expansion and/or survival of tdTom+ population. Notably, RUNX1c/tdTom+ cells represent only a limited subpopuation of CD34+CD45+ and CD34+CD43+ cells with a unique genetic signature. Using gene array analysis, we find significantly lower expression of Let-7 and mir181a microRNAs in the RUNX1c/tdTom+ cell population. These phenotypic and genetic analyses comparing the RUNX1c/tdTom+ population to CD34+CD45+ umbilical cord blood and fetal liver demonstrate several key differences that likely impact the development of HSCs capable of long-term multilineage engraftment from hESCs and iPSCs.

347 Both human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) are pluripotent stem cells (hPSCs) with potential to differentiate into all types of somatic cells. Patients suffering from blood disorders can be cured with hematopoietic cell transplantations (HCT). Technical advancements in hPSC production and handling have revolutionized their potential applications in regenerative medicine and provided enormous hope for patients who may need HCT. hiPSCs derived from autologous cells could provide unlimited leukocyte antigen matched blood cells on a patient-specific basis. A remaining hurdle in this process remains the need for efficient and effective generation of specific blood cells from hPSCs for therapeutic use. Transcription factors play key roles in regulating maintenance, expansion, and differentiation of blood cells from hPSCs. Studies have shown that transcription factor RUNX1 is required for the formation of definitive blood cells. There are several alternatively spliced isoforms of the RUNX1 protein, including the shortest form RUNX1a and two longer forms RUNX1b and RUNX1c. Based on known properties of RUNX1 proteins, we hypothesized that RUNX1a promotes the production of therapeutic hematopoietic stem cells from hPSCs. By employing ectopic expression of RUNX1a on different human ESC and iPSC lines (H9, BC1, iCB5) under a defined hematopoietic differentiation system, we aimed to identify function of RUNX1a on lineage commitment and molecular mechanisms of RUNX1 activity in differentiation of PSCs to hematopoietic cells. We demonstrated that expression of endogenous RUNX1a parallels lineage commitment and hematopoietic emergence from hPSCs. During differentiation process RUNX1a enhanced the expression of several mesoderm and hematopoietic differentiation related factors, including KDR, SCL, GATA2, and PU.1. In addition, over-expression of RUNX1a in embryoid bodies (EBs) showed more efficient and earlier emergence of typical sac structures, which predicts cell lineage commitment and germ layer development at the early stage of EB differentiation. At day 7, EBs derived from hPSCs was dissociated into single cells for flow cytometry analysis. The mean frequency of CD31+CD34+CD45− and total CD34+ cells with hemato-endothelial cell features are 35.1% and 67.1% from RUNX1a-overexpressing EBs, and 8.7% and 24.1% from vector control EBs. Immunohistochemistry analysis of EBs at day 9 of differentiation confirmed that expression of RUNX1a accelerated mesoderm commitment and emergence of hemato-endothelial precursors. Flow cytometry analysis on EBs collected at days 9, 11, 13 showed that ectopic RUNX1a induced a robust increase in the frequency of hematopoietic progenitor cells in all hPSC lines examined. At Day 9, RUNX1a-overexpressing EBs generated 48.5% CD43+CD45+ cells, 45.1% CD34+CD45+ cells, and 8.5 folds higher CD43+ cells than vector EBs. Later at Day 13, 80% CD45+ and 75% CD43+/CD34+CD45+ hematopoietic stem/progenitor cells (HSPCs) achieved from dissociated EBs. In liquid culture, RUNX1a HSPC showed strong expansion and high percentage of CD235a+CD45− (20%) and CD71+CD235a+ (16%), markers for erythroid populations. Flow cytometry and western blots on RUNX1a-EB formed colonies showed significantly higher β-globin production than that of the vector, suggesting expression of RUNX1a in HSPC enhanced definitive hematopoiesis. RUNX1a-hPSCs derived HSPCs possess self-renewal capability and are capable of differentiating into multi-lineages ex vivo. Furthermore HSPCs generated from RUNX1a-EBs possessed the capacity of interacting with surrogate niche and showed long-term repopulation ability under LTC-IC (Long-Term Culture-Initiating Cell Assay) condition. Colonies generated from HSPC of RUNX1a-EBs after 3 week bulk LTC-IC culture showed 300 folds higher than vector control. RUNX1a-hPSCs derived CD34+CD45+ cells could maintain a non-adherent population in ouldCD45+ sEBsND THIS SENTENCE5 week culture on stromal cell M210. In summary we identified that RUNX1a enhances derivation of definitive hematopoietic cells from human PSCs. Our study provides an important and useful system to enhance specificity and efficiency of generating functional blood cells and further differentiated cells from human PSCs, which may provide valuable source for future clinical applications in patients with hematologic disorders. Disclosures: No relevant conflicts of interest to declare.

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.