Sandra Capellera-Garcia

Abstract Hematopoietic stem cells (HSCs) derived from human induced pluripotent stem cells (iPS cells) have important biomedical applications. We identified differentiation conditions that generate HSCs defined by robust long-term multilineage engraftment in immune-deficient NOD,B6. Prkdc scid Il2rg tm1Wjl/SzJ Kit W41/W41 mice. We guided differentiating iPS cells, as embryoid bodies in a defined culture medium supplemented with retinyl acetate, through HOXA -patterned mesoderm to hemogenic endothelium specified by bone morphogenetic protein 4 and vascular endothelial growth factor (VEGF). Removal of VEGF facilitated an efficient endothelial-to-hematopoietic transition, evidenced by release into the culture medium of CD34 + blood cells, which were cryopreserved. Intravenous transplantation of two million thawed CD34 + cells differentiated from four independent iPS cell lines produced multilineage bone marrow engraftment in 25–50% of immune-deficient recipient mice. These functionally defined, multipotent CD34 + hematopoietic cells, designated iPS cell-derived HSCs (iHSCs), produced levels of engraftment similar to those achieved following umbilical cord blood transplantation. Our study provides a step toward the goal of generating HSCs for clinical translation.

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of well characterized genes. However, the minimal set of factors necessary for instructing red blood cell (RBC) development remains undefined. We employed a screen for transcription factors allowing direct lineage reprograming from fibroblasts to induced erythroid progenitors/precursors (iEPs). We show that Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs. The transcriptional signature of murine iEPs resembled mainly that of primitive erythroid progenitors in the yolk sac, whereas addition of Klf1 or Myb to the GTLM cocktail resulted in iEPs with a more adult-type globin expression pattern. Our results demonstrate that direct lineage conversion is a suitable platform for defining and studying the core factors inducing the different waves of erythroid development.

, display polylobulated nuclei, have ploidies higher than 4N, form MK colonies, and give rise to platelets in vitro. Moreover, transplantation of MK-like murine progenitor cells into NSG mice results in successful engraftment and further maturation in vivo. Similar results are obtained using disease-corrected fibroblasts from Fanconi anemia patients. Our results combined demonstrate that functional MK progenitors with clinical potential can be obtained in vitro, circumventing the use of hematopoietic progenitors or pluripotent stem cells.

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.