Direct Conversion of Skin Cells into Blood: Alchemy or Science?
Authors: Katherine Y King, Margaret A Goodell
Journal: Molecular Therapy (2011)

Our neighborhood blood bank just announced an emergency Thanksgiving-season blood drive. For years, the field of stem cell biology has described an elusive but commonly understood goal: the elimination of a need for blood drives. Many years of work have led to methods for generation of bona fide hematopoietic cells from human and mouse embryonic stem cells, but problems with efficiency as well as maturation of the blood cells persist, impeding the utility of these methods in manufacturing blood. Now, in the era of “direct reprogramming,” we can imagine a goal of converting an adult cell, such as a skin cell, into a hematopoietic stem cell that can be used for autologous blood replacement (Figure 1). In an article recently published in Nature, Szabo et al. report a huge step toward that goal.1Szabo E Rampalli S Risueño RM Schnerch A Mitchell R Fiebig-Comyn A et al.Direct conversion of human fibroblasts to multilineage blood progenitors.Nature. 2010; 468: 521-526Crossref PubMed Scopus (600) Google Scholar The authors describe the generation of differentiated hematopoietic cells following the enforced expression of the POU-domain transcription factor OCT4 in adult or neonatal human skin fibroblasts. The alchemy of turning a person's skin into blood has so far been elusive. Work by Jaenisch, Yamanaka, and others has suggested that the properties of differentiated adult cells must be “dialed back” to recreate the status of a pluripotent stem cell before redifferentiation into another cell type such as a hematopoietic progenitor cell. Those reprogramming steps have generally involved the forced expression of pluripotency factors and their transcriptional programs: OCT4, KLF4, and cMYC.2Okita K Nakagawa M Hyenjong H Ichisaka T Yamanaka S Generation of mouse induced pluripotent stem cells without viral vectors.Science. 2008; 322: 949-953Crossref PubMed Scopus (1622) Google Scholar Combined exogenous expression of these factors can confer the characteristics of pluripotent stem cells on a differentiated adult cell such as a fibroblast. A major stumbling block in this effort has been that cells subjected to forced expression of such pluripotency factors can become transformed, leading to unrestricted dysplastic growth and failure to differentiate into mature blood cells.3Hochedlinger K Yamada Y Beard C Jaenisch R Ectopic expression of Oct-4 blocks progenitor-cell differentiation and causes dysplasia in epithelial tissues.Cell. 2005; 121: 465-477Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar Another approach to the generation of blood replacement has been to enhance the ability of embryonic stem cells to differentiate into hematopoietic cells. Kyba et al. overexpressed the homeobox gene, HoxB4, in embryoid body–derived cells to generate hematopoietic progenitors characterized by long-term myeloid and lymphoid engraftment potential (i.e., the ability to make the major blood cell lineages, including macrophages and lymphocytes). This approach, although relatively successful, was complicated by an uneven capacity to differentiate into all blood lineages and the generation of a pre-transformed state at risk for development of leukemia.4Kyba M Perlingeiro RC Daley GQ HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors.Cell. 2002; 109: 29-37Abstract Full Text Full Text PDF PubMed Scopus (653) Google Scholar Szabo et al. now report some true-life alchemy—enforced expression of the POU-domain transcription factor OCT4, alone in adult or neonatal human skin fibroblasts, led to the generation of differentiated hematopoietic cells, while entirely bypassing a pluripotent state.1Szabo E Rampalli S Risueño RM Schnerch A Mitchell R Fiebig-Comyn A et al.Direct conversion of human fibroblasts to multilineage blood progenitors.Nature. 2010; 468: 521-526Crossref PubMed Scopus (600) Google Scholar OCT4 expression appeared to drive the emergence of CD45+ hematopoietic cells at a whopping 38% of transduced cells, and these cells could support growth of hematopoietic colonies. In humans, hematopoietic stem cells express the cell surface marker CD34. These findings are particularly striking given that prior efforts at cellular reprogramming have required the concurrent expression of multiple transcription factors, not just one. The challenge of transfecting cells with three or more independent viral expression vectors, and the still poorly understood reprogramming process, generally results in an efficiency of reprogramming of less than 0.01% of transduced cells.2Okita K Nakagawa M Hyenjong H Ichisaka T Yamanaka S Generation of mouse induced pluripotent stem cells without viral vectors.Science. 2008; 322: 949-953Crossref PubMed Scopus (1622) Google Scholar Here, the reported high efficiency of reprogramming, albeit directly to blood cells rather than to pluripotent cells, with transduction by a single factor, dramatically increases the practicality of the approach in principle. Following culture in the appropriate cytokine milieu, the OCT4-overexpressing cells could support myeloid differentiation; in the presence of a mixture of six cytokine growth factors, the cells could be induced to form monocytes. With the addition of two more factors, the cells differentiated into macrophages that had the ability to engulf green fluorescent protein–labeled latex beads. In the presence of erythropoietin, the cells could be driven to express the adult isoform of β-globin, reflecting full differentiation of definitive hematopoietic cells from fetal-type undifferentiated progenitors. Furthermore, these cells took on characteristics of mature megakaryocytes. These findings indicate that the OCT4-expressing fibroblasts could give rise to at least two types of differentiated blood cells within the myeloid lineage: macrophages and megakaryocytes. The gold-standard test for a hematopoietic stem cell is transplantation, in which the cells under investigation are tested for their ability to fully reconstitute all lineages of the peripheral blood in a lethally irradiated, bone marrow–depleted mouse. In this study, Szabo et al. demonstrated that xenotransplantation of OCT4-expressing fibroblasts into immunodeficient mice led to primary engraftment, indicating hematopoietic progenitor capability in vivo.1Szabo E Rampalli S Risueño RM Schnerch A Mitchell R Fiebig-Comyn A et al.Direct conversion of human fibroblasts to multilineage blood progenitors.Nature. 2010; 468: 521-526Crossref PubMed Scopus (600) Google Scholar The authors did not assess the production of all blood cell lineages in the transplanted mice, noting only that there was a myeloid predominance and hence relative lack of lymphocytes. Without evidence for lymphoid development, we lack the information to determine whether these cells meet the criteria for hematopoietic stemness. Nonetheless, demonstration of myeloid hematopoietic potential in these transplanted mice in vivo is exciting indeed. Importantly, ectopic expression of OCT4 did not drive expression of pluripotency gene signatures or canonical pluripotency markers, including SSEA3 and Tra-1-60. These findings are strengthened by chromatin immunoprecipitation data showing that OCT4 bound to genes characteristically expressed in hematopoietic stem cells (RUNX1, PU.1, C/EBPa, SCL-TAL1) but not to pluripotency markers that are expressed in embryonic stem cells (SOX2, NANOG). Thus, OCT4 appeared to specifically activate a hematopoietic program of pluripotency but not the more primitive transcriptional programs of embryonic stem cells. In addition, whereas induced pluripotent stem cells, like embryonic stem cells, tend to form teratomas, OCT4-transduced dermal fibroblasts showed no propensity for immortalization. These findings are particularly significant, given that transformation into leukemogenic cells has been a major concern when using embryonic stem cells to generate hematopoietic cells. Surprisingly, incubation of the OCT4-transduced fibroblasts in medium containing the cytokine erythropoietin led to emergence of megakaryocyte progenitors, which give rise to platelets, but not erythroid cells such as red blood cells. Indeed, neither erythroid nor lymphoid cells developed from the transduced cells despite expression of CD34, a hematopoietic stem cell marker, on fully one-quarter of the transduced cells. Given the vast prior literature showing that OCT4 is a pluripotency factor specifically for embryonic-like pluripotential (and hence highly undifferentiated) stem cells and, furthermore, that mice with somatic mutations in OCT4 have no defect in adult bone marrow function, it is particularly surprising that OCT4 expression alone would support myeloid-specific differentiation.5Lengner CJ Camargo FD Hochedlinger K Welstead GG Zaidi S Gokhale S et al.Oct4 expression is not required for mouse somatic stem cell self-renewal.Cell Stem Cell. 2007; 1: 403-415Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar,6Lengner CJ Welstead GG Jaenisch R The pluripotency regulator Oct4: a role in somatic stem cells?.Cell Cycle. 2008; 7: 725-728Crossref PubMed Scopus (99) Google Scholar It is possible that OCT4 overexpression imposes a differentiation program not seen at the level of expression normally observed in embryonic stem cells. Alternatively, this differentiation bias may be a property of human but not mouse OCT4. The development of hematopoietic cell characteristics from fibroblasts was dependent on exposure of the cells to a prohematopoietic cytokine mixture including insulin-like growth factor-2, basic fibroblast growth factor, stem cell factor, and fms-related tyrosine kinase-3 ligand. Unfortunately, some of the experiments described in Szabo and colleagues' article omitted the critical control of untransduced fibroblasts exposed to the cytokine mixture. Because the purity of the fibroblasts is not described, this leaves open the possibility that peripherally located hematopoietic stem cells or dendritic cells were present in the fibroblast cell culture and were amplified by the culture conditions. This possibility might also explain the singular differentiation potential of the transduced fibroblasts to form myeloid lineage cells. Nonetheless, Szabo and colleagues' data are tantalizing. The high efficiency of generation of hematopoietic cells from the transduced cells and the avoidance of transcriptional pathways linked to pluripotency are critical steps toward achieving autologous hematopoietic cell replacement therapy. It will be interesting to see how these results play out in future studies. In the meantime, we will still be collecting our free T-shirts at the blood drive next week.