Differential Effects of HOXB4 on Nonhuman Primate Short- and Long-Term Repopulating Cells

BACKGROUND: Hematopoietic stem cells (HSCs) or repopulating cells are able to self-renew and differentiate into cells of all hematopoietic lineages, and they can be enriched using the CD34 cell surface marker. Because of this unique property, HSCs have been used for HSC transplantation and gene therapy applications. However, the inability to expand HSCs has been a significant limitation for clinical applications. Here we examine, in a clinically relevant nonhuman primate model, the ability of HOXB4 to expand HSCs to potentially overcome this limitation. METHODS AND FINDINGS: Using a competitive repopulation assay, we directly compared in six animals engraftment of HOXB4GFP (HOXB4 green fluorescent protein) and control (yellow fluorescent protein [YFP])–transduced and expanded CD34 (+) cells. In three animals, cells were infused after a 3-d transduction culture, while in three other animals cells were infused after an additional 6–9 d of ex vivo expansion. We demonstrate that HOXB4 overexpression resulted in superior engraftment in all animals. The most dramatic effect of HOXB4 was observed early after transplantation, resulting in an up to 56-fold higher engraftment compared to the control cells. At 6 mo after transplantation, the proportion of marker gene–expressing cells in peripheral blood was still up to 5-fold higher for HOXB4GFP compared to YFP-transduced cells. CONCLUSIONS: These data demonstrate that HOXB4 overexpression in CD34 (+) cells has a dramatic effect on expansion and engraftment of short-term repopulating cells and a significant, but less pronounced, effect on long-term repopulating cells. These data should have important implications for the expansion and transplantation of HSCs, in particular for cord blood transplantations where often only suboptimal numbers of HSCs are available

ES-Cell Derived Hematopoietic Cells Induce Transplantation Tolerance

Background: Bone marrow cells induce stable mixed chimerism under appropriate conditioning of the host, mediating the induction of transplantation tolerance. However, their strong immunogenicity precludes routine use in clinical transplantation due to the need for harsh preconditioning and the requirement for toxic immunosuppression to prevent rejection and graft-versus-host disease. Alternatively, embryonic stem (ES) cells have emerged as a potential source of less immunogenic hematopoietic progenitor cells (HPCs). Up till now, however, it has been difficult to generate stable hematopoietic cells from ES cells. Methodology/Principal Findings: Here, we derived CD45 + HPCs from HOXB4-transduced ES cells and showed that they poorly express MHC antigens. This property allowed their long-term engraftment in sublethally irradiated recipients across MHC barriers without the need for immunosuppressive agents. Although donor cells declined in peripheral blood over 2 months, low level chimerism was maintained in the bone marrow of these mice over 100 days. More importantly, chimeric animals were protected from rejection of donor-type cardiac allografts. Conclusions: Our data show, for the first time, the efficacy of ES-derived CD45 + HPCs to engraft in allogenic recipient

Concise review:programming human pluripotent stem cells into blood

Blood disorders are treated with cell therapies including haematopoietic stem cell (HSC) transplantation as well as platelet and red blood cell transfusions. However the source of cells is entirely dependent on donors, procedures are susceptible to transfusion‐transmitted infections and serious complications can arise in recipients due to immunological incompatibility. These problems could be alleviated if it was possible to produce haematopoietic cells in vitro from an autologous and renewable cell source. The production of haematopoietic cells in the laboratory from human induced pluripotent stem cells (iPSCs) may provide a route to realize this goal but it has proven challenging to generate long‐term reconstituting HSCs. To date, the optimization of differentiation protocols has mostly relied on the manipulation of extrinsic signals to mimic the in vivo environment. We review studies that have taken an alternative approach to modulate intrinsic signals by enforced expression of transcription factors. Single and combinations of multiple transcription factors have been used in a variety of contexts to enhance the production of haematopoietic cells from human pluripotent stem cells. This programming approach, together with the recent advances in the production and use of synthetic transcription factors, holds great promise for the production of fully functional HSCs in the future

The HOXB4 Homeoprotein Promotes the Ex Vivo Enrichment of Functional Human Embryonic Stem Cell-Derived NK Cells

Human embryonic stem cells (hESCs) can be induced to differentiate into blood cells using either co-culture with stromal cells or following human embryoid bodies (hEBs) formation. It is now well established that the HOXB4 homeoprotein promotes the expansion of human adult hematopoietic stem cells (HSCs) but also myeloid and lymphoid progenitors. However, the role of HOXB4 in the development of hematopoietic cells from hESCs and particularly in the generation of hESC-derived NK-progenitor cells remains elusive. Based on the ability of HOXB4 to passively enter hematopoietic cells in a system that comprises a co-culture with the MS-5/SP-HOXB4 stromal cells, we provide evidence that HOXB4 delivery promotes the enrichment of hEB-derived precursors that could differentiate into fully mature and functional NK. These hEB-derived NK cells enriched by HOXB4 were characterized according to their CMH class I receptor expression, their cytotoxic arsenal, their expression of IFNγ and CD107a after stimulation and their lytic activity. Furthermore our study provides new insights into the gene expression profile of hEB-derived cells exposed to HOXB4 and shows the emergence of CD34+CD45RA+ precursors from hEBs indicating the lymphoid specification of hESC-derived hematopoietic precursors. Altogether, our results outline the effects of HOXB4 in combination with stromal cells in the development of NK cells from hESCs and suggest the potential use of HOXB4 protein for NK-cell enrichment from pluripotent stem cells

Epigenetic memory in induced pluripotent stem cells

Somatic cell nuclear transfer and transcription-factor-based reprogramming revert adult cells to an embryonic state, and yield pluripotent stem cells that can generate all tissues. Through different mechanisms and kinetics, these two reprogramming methods reset genomic methylation, an epigenetic modification of DNA that influences gene expression, leading us to hypothesize that the resulting pluripotent stem cells might have different properties. Here we observe that low-passage induced pluripotent stem cells (iPSCs) derived by factor-based reprogramming of adult murine tissues harbour residual DNA methylation signatures characteristic of their somatic tissue of origin, which favours their differentiation along lineages related to the donor cell, while restricting alternative cell fates. Such an ‘epigenetic memory’ of the donor tissue could be reset by differentiation and serial reprogramming, or by treatment of iPSCs with chromatin-modifying drugs. In contrast, the differentiation and methylation of nuclear-transfer-derived pluripotent stem cells were more similar to classical embryonic stem cells than were iPSCs. Our data indicate that nuclear transfer is more effective at establishing the ground state of pluripotency than factor-based reprogramming, which can leave an epigenetic memory of the tissue of origin that may influence efforts at directed differentiation for applications in disease modelling or treatment.National Institutes of Health (U.S.) (NIH grant RO1-DK70055)National Institutes of Health (U.S.) (NIH Grant RO1-DK59279)National Institutes of Health (U.S.) (American Recovery and Reinvestment Act (RC2-HL102815))National Institutes of Health (U.S.) (NIH (K99HL093212-01))Cooley’s Anemia FoundationNational Institutes of Health (U.S.) (NIH LLS (3567-07))National Institutes of Health (U.S.) (NIH grant R37CA054358)National Institutes of Health (U.S.) (NIH grant P50HG003233)National Institutes of Health (U.S.) (NIH grant R01AI047457)National Institutes of Health (U.S.) (NIH Grant R01AI047458)National Institutes of Health (U.S.) (CA86065)National Institutes of Health (U.S.) (HL099999)Thomas and Stacey Siebel FoundationCalifornia Institute for Regenerative Medicine (Fellowship T1-00001

Evolution of reproductive development in the volvocine algae

The evolution of multicellularity, the separation of germline cells from sterile somatic cells, and the generation of a male–female dichotomy are certainly among the greatest innovations of eukaryotes. Remarkably, phylogenetic analysis suggests that the shift from simple to complex, differentiated multicellularity was not a unique progression in the evolution of life, but in fact a quite frequent event. The spheroidal green alga Volvox and its close relatives, the volvocine algae, span the full range of organizational complexity, from unicellular and colonial genera to multicellular genera with a full germ–soma division of labor and male–female dichotomy; thus, these algae are ideal model organisms for addressing fundamental issues related to the transition to multicellularity and for discovering universal rules that characterize this transition. Of all living species, Volvox carteri represents the simplest version of an immortal germline producing specialized somatic cells. This cellular specialization involved the emergence of mortality and the production of the first dead ancestors in the evolution of this lineage. Volvocine algae therefore exemplify the evolution of cellular cooperation from cellular autonomy. They also serve as a prime example of the evolution of complex traits by a few successive, small steps. Thus, we learn from volvocine algae that the evolutionary transition to complex, multicellular life is probably much easier to achieve than is commonly believed

An inducible arylsulfatase of Volvox carteri with properties suitable for a reporter-gene system. Purification, characterization and molecular cloning

The multicellular green flagellate Volvox carteri synthesizes a periplasmic arylsulfatase in response to sulfur deprivation. The inducible enzyme has been purified to homogeneity and characterized. The corresponding gene and cDNA have been cloned. Determination of the sequence of genomic clones and comparisons to the cDNA sequence, revealed sixteen introns and seventeen exons that encode a 649-amino-acid polypeptide chain. Since the arylsulfatase enzyme is readily assayed using chromogenic substrates, but is not detectable in cells grown in sulfate-containing medium, the gene encoding arylsulfatase may be useful as a reporter gene in V. carteri. In addition, the highly regulated promoter of the arylsulfatase gene suggests its suitability as a tool for producing inducible expression vectors for cloned genes