Efficient generation, purification, and expansion of CD34(+) hematopoietic progenitor cells from nonhuman primate-induced pluripotent stem cells.
ABSTRACT: Induced pluripotent stem cell (iPSC) therapeutics are a promising treatment for genetic and infectious diseases. To assess engraftment, risk of neoplastic formation, and therapeutic benefit in an autologous setting, testing iPSC therapeutics in an appropriate model, such as the pigtail macaque (Macaca nemestrina; Mn), is crucial. Here, we developed a chemically defined, scalable, and reproducible specification protocol with bone morphogenetic protein 4, prostaglandin-E2 (PGE2), and StemRegenin 1 (SR1) for hematopoietic differentiation of Mn iPSCs. Sequential coculture with bone morphogenetic protein 4, PGE2, and SR1 led to robust Mn iPSC hematopoietic progenitor cell formation. The combination of PGE2 and SR1 increased CD34(+)CD38(-)Thy1(+)CD45RA(-)CD49f(+) cell yield by 6-fold. CD34(+)CD38(-)Thy1(+)CD45RA(-)CD49f(+) cells isolated on the basis of CD34 expression and cultured in SR1 expanded 3-fold and maintained this long-term repopulating HSC phenotype. Purified CD34(high) cells exhibited 4-fold greater hematopoietic colony-forming potential compared with unsorted hematopoietic progenitors and had bilineage differentiation potential. On the basis of these studies, we calculated the cell yields that must be achieved at each stage to meet a threshold CD34(+) cell dose that is required for engraftment in the pigtail macaque. Our protocol will support scale-up and testing of iPSC-derived CD34(high) cell therapies in a clinically relevant nonhuman primate model.
Project description:Induced pluripotent stem cells (iPSCs) offer a promising platform to model early embryonic developmental processes, to create disease models that can be evaluated by drug screens as well as proof-of-concept experiments for regenerative medicine. However, generation of iPSC-derived hemato-endothelial and hematopoietic progenitor cells for these applications is challenging due to variable and limited cell numbers, which necessitates enormous up-scaling or development of demanding protocols. Here, we unravel the function of key transcriptional regulators SCL, LMO2, GATA2, and ETV2 (SLGE) on early hemato-endothelial specification and establish a fully inducible and stepwise hemato-endothelial forward programming system based on SLGE-regulated overexpression. Regulated induction of SLGE in stable SLGE-iPSC lines drives very efficient generation of large numbers of hemato-endothelial progenitor cells (CD144+/CD73-), which produce hematopoietic progenitor cells (CD45+/CD34+/CD38-/CD45RA-/CD90+/CD49f+) through a gradual process of endothelial-to-hematopoietic transition (EHT).
Project description:Gene expression profiling was used to investigate the relationship between human cord blood hematopoietic stem cells (HSC) and multipotent progenitors described in the study. Genes more highly expressed in HSC may be associated with stem cell function and self-renewal. Total RNA was obtained from flow-sorted populations based on the cell surface expression of CD34, CD38, CD45RA, Thy1, and CD49f.
Project description:Gene expression profiling was used to investigate the relationship between human cord blood hematopoietic stem cells (HSC) and multipotent progenitors described in the study. Genes more highly expressed in HSC may be associated with stem cell function and self-renewal. Overall design: Total RNA was obtained from flow-sorted populations based on the cell surface expression of CD34, CD38, CD45RA, Thy1, and CD49f.
Project description:Priming haematopoietic stem/progenitor cells (HSPCs) in vitro with specific chromatin modifying agents and cytokines under serum-free-conditions significantly enhances engraftable HSC numbers. We extend these studies by culturing human CD133+ HSPCs on nanofibre scaffolds to mimic the niche for 5-days with the HDAC inhibitor Scriptaid and cytokines. Scriptaid increases absolute Lin-CD34+CD38-CD45RA-CD90+CD49f+ HSPC numbers, while concomitantly decreasing the Lin-CD38-CD34+CD45RA-CD90- subset. Hypothesising that Scriptaid plus cytokines expands the CD90+ subset without differentiation and upregulates CD90 on CD90- cells, we sorted, then cultured Lin-CD34+CD38-CD45RA-CD90- cells with Scriptaid and cytokines. Within 2-days and for at least 5-days, most CD90- cells became CD90+. There was no significant difference in the transcriptomic profile, by RNAsequencing, between cytokine-expanded and purified Lin-CD34+CD38-CD45RA-CD49f+CD90+ cells in the presence or absence of Scriptaid, suggesting that Scriptaid maintains stem cell gene expression programs despite expansion in HSC numbers. Supporting this, 50 genes were significantly differentially expressed between CD90+ and CD90- Lin-CD34+CD38-CD45RA-CD49f+ subsets in Scriptaid-cytokine- and cytokine only-expansion conditions. Thus, Scriptaid treatment of CD133+ cells may be a useful approach to expanding the absolute number of CD90+ HSC, without losing their stem cell characteristics, both through direct effects on HSC and potentially also conversion of their immediate CD90- progeny into CD90+ HSC.
Project description:Transplantation of ex vivo expanded human umbilical cord blood cells (hCB) only partially enhances the hematopoietic recovery after myelosuppressive therapy. Incubation of hCB with optimal combinations of cytokines and niche cells, such as endothelial cells (ECs), could augment the efficiency of hCB expansion. We have devised an approach to cultivate primary human ECs (hECs) in serum-free culture conditions. We demonstrate that coculture of CD34(+) hCB in direct cellular contact with hECs and minimal concentrations of thrombopoietin/Kit-ligand/Flt3-ligand resulted in a 400-fold expansion of total hematopoietic cells, 150-fold expansion of CD45(+)CD34(+) progenitor cells, and 23-fold expansion of CD45(+) Lin(-)CD34(hi+)CD45RA(-)CD49f(+) stem and progenitor cells over a 12-day period. Compared with cytokines alone, coculture of hCB with hECs permitted greater expansion of cells capable of multilineage engraftment and serial transplantation, hallmarks of long-term repopulating hematopoietic stem cells. Therefore, hECs establish a cellular platform for expansion of hematopoietic stem and progenitor cells and treatment of hematologic disorders.
Project description:Here we tracked transcriptome changes over ten human hematopoietic stem and early progenitor populations, defining the transcriptional dynamics underlying the first steps of commitment. Most of the transcriptional programs observed extended beyond lineage boundaries. In particular, multi-lymphoid progenitors (MLPs) presented a hybrid transcriptional state with elements of lymphoid and myeloid programs, but also stem cell characteristics. Total RNA was obtained from flow-sorted populations of human cord blood based on the cell surface expression of CD34, CD38, CD45RA, Thy1, and CD49f, CD10, CD7, CD19 and CD1a.
Project description:Human embryonic stem cell (hESC)-derived hematopoietic stem/progenitor cells hold tremendous potential as alternative cell sources for the treatment of various hematological diseases, drug discovery and toxicological screening. However, limited number of hematopoietic stem/progenitor cells generated from the differentiation of hESCs hinders their downstream applications. Here, we show that aryl hydrocarbon receptor antagonist StemRegenin 1 (SR1) selectively promotes expansion of hESC-derived lin-CD34+ hematopoietic progenitors in a concentration-dependent manner. The colony-forming cell (CFC) activity was found to be enriched in the CD34+ cells that were expanded with SR1; however, these cells have less colony-forming activity as compared to unexpanded cells (1,338 vs. 7 of CD34+ cells to form 1 colony, respectively). Interestingly, SR1 showed a bipotential effect on the proliferation of CD34 negative population, that is low dose of SR1 (1 µM) enhanced cell proliferation, whereas it was repressed at higher doses (>5 µM). In summary, our results suggest that SR1 has the potential to facilitate expansion of hESC-derived lin-CD34+ hematopoietic progenitors, which further retain the potential to form multilineage hematopoietic colonies.
Project description:<h4>Background</h4>Ex vivo production of hematopoietic stem/precursor cells (HSPCs) represents a promising versatile approach for blood disorders.<h4>Methods</h4>To derive definitive HSPCs from human embryonic stem cells (ESCs), we differentiated mesodermally specified embryoid bodies (EBs) on gelatin-coated plates in serum/feeder-free conditions.<h4>Results</h4>Seven-day EB maturation followed by an 8-day differentiation period on OP9 cells provided the highest number of definitive (CD34+ CD235a-, 69%, p?<?0.01) and lowest number of primitive (CD34- CD235a+, 1.55%, p?<?0.01) precursor cells along with the highest colony-forming units (149.8?±?11.6, p?<?0.01) in feeder-free conditions. Maximal HSPC fraction (CD34+ CD38- CD45RA- CD49f+ CD90+) was 7.6-8.9% after 10?days of hematopoietic differentiation with 14.5% adult ?-globin expression following RBC differentiation. Myeloid and erythroid colonies were restricted strictly to the CD34+ CD43+ fraction (370.5?±?65.7, p?<?0.001), while the CD34- CD43+ fraction produced only a small number of colonies (21.6?±?11.9). In addition, we differentiated the CD34+ CD43+ cells towards T-lymphocytes using the OP9/DLL1 co-culture system demonstrating double-positive T cells (CD4+ CD8+) with CD3+ expression displaying a broad T cell receptor (TCR) repertoire. Confocal imaging of organoid-like structures revealed a close association of CD31+ cells with CD34+ and CD43+ cells, suggesting a potential emergence of HSPCs through endothelial to hematopoietic transition. Furthermore, fluorescently labeled organoids exhibited the emergence of spherical non-attached cells from rare progenitors at the border of the organoid center.<h4>Conclusions</h4>In summary, definitive HSPCs can be derived from ESCs through a dynamic cellular process from an organoid-like structure, where erythroid progeny are capable of producing adult hemoglobin and lymphoid progeny shows a diverse TCR repertoire.
Project description:The main limitations of hematopoietic cord blood (CB) transplantation, viz, low cell dosage and delayed reconstitution, can be overcome by ex vivo expansion. CB expansion under conventional culture causes rapid cell differentiation and depletion of hematopoietic stem and progenitor cells (HSPCs) responsible for engraftment. In this study, we use combinatorial cell culture technology (CombiCult<sup>®</sup>) to identify medium formulations that promote CD133<sup>+</sup> CB HSPC proliferation while maintaining their phenotypic characteristics. We employed second-generation CombiCult screens that use electrospraying technology to encapsulate CB cells in alginate beads. Our results suggest that not only the combination but also the order of addition of individual components has a profound influence on expansion of specific HSPC populations. Top protocols identified by the CombiCult screen were used to culture human CD133<sup>+</sup> CB HSPCs on nanofiber scaffolds and validate the expansion of the phenotypically defined CD34<sup>+</sup>CD38<sup>lo/-</sup>CD45RA<sup>-</sup>CD90<sup>+</sup>CD49f<sup>+</sup> population of hematopoietic stem cells and their differentiation into defined progeny.