diffi culty in the generation of core-shell NPs with a lipid shell containing various amounts of water, which governs the rigidity of the NPs; larger amounts of interfacial water would result in more fl exible NPs. [13][14][15] Microfl uidic platforms can generate lipid-polymer hybrid NPs via rapid reaction and precise manipulation of fl uids inside microchannels; [16][17][18][19][20] however, the fabrication of hybrid NPs with varying water content has not been achieved by microfl uidics. Here, we develop a two-stage microfl uidic platform that can assemble core-shell poly(lactic-co -glycolic acid) (PLGA)-lipid NPs in a single-step. [ 16,21 ] Lipid-covered PLGA NPs or liposomes that have the same size and surface properties, but varying rigidity as a result of tuning the interfacial water layer, can be realized using the same microchip. It enables us to explore how the rigidity of NPs differentially regulates the cellular uptake and to elucidate the intrinsic mechanism. It also allows the treatment of various diseases through the use of specifi c particles.Particle rigidity is tuned by varying the amounts of interfacial water between the PLGA core and lipid shell of the hybrid NPs; this is achieved by altering the injection order of the PLGA and lipid-poly(ethylene glycol) (PEG) organic solutions in the microfl uidic chip. The microfl uidic device shown in Scheme 1 consists of two stages: 1) The fi rst stage comprises three inlets and a straight synthesis microchannel; 2) The second stage is composed of one centered inlet and a spiral mixing channel (see Supporting Information (SI), Figure S1 for more details). We synthesized particles of varying water content and rigidity using the same chip but different order of the introducing reagents. In mode 1, the fi rst stage is used for generating PLGA NPs through interfacial precipitation, while the second stage forms lipid-coated NPs as a result of hydrophobic attraction between the lipid tail and PLGA (P-L NPs; Scheme 1 A, Figure S2 (SI)). In mode 2, we change the injection order by introducing the lipid solution at the fi rst stage and the PLGA solution at the second stage. In this way, lipids form into a liposome in aqueous solution at the fi rst stage, followed by re-assembly onto the surface of PLGA NPs at the second stage through effective mixing (P-W-L NPs; Scheme 1 B, Figure S2 (SI)). The throughput of NPs by a single chip is 41 mL h −1 (≈8 mg h −1 for P-W-L NPs, and ≈6.5 mg h −1 for P-L NPs). For both mode 1 and mode 2, transmission electron microscopy (TEM) images ( Figure 1 A; Figure S2, SI) show complete lipid coverage on the surface of PLGA NPs. The different injection order of the solutions may result in the presence of interfacial water between the PLGA core and lipid shell of the P-W-L NPs (mode 2) but not in P-L NPs (mode 1), which is confi rmed by cryogenic TEM (cryo-TEM Figure 1 B; see also SI). For the P-L NPs, the lipid shell is tightly attached to the PLGA core, while for the P-W-L Even though much research has shown that nanoparticles (NPs) ca...

SummaryHuman induced pluripotent stem cells (iPSCs) provide a potentially replenishable source for the production of transfusable platelets. Here, we describe a method to generate megakaryocytes (MKs) and functional platelets from iPSCs in a scalable manner under serum/feeder-free conditions. The method also permits the cryopreservation of MK progenitors, enabling a rapid “surge” capacity when large numbers of platelets are needed. Ultrastructural/morphological analyses show no major differences between iPSC platelets and human blood platelets. iPSC platelets form aggregates, lamellipodia, and filopodia after activation and circulate in macrophage-depleted animals and incorporate into developing mouse thrombi in a manner identical to human platelets. By knocking out the β2-microglobulin gene, we have generated platelets that are negative for the major histocompatibility antigens. The scalable generation of HLA-ABC-negative platelets from a renewable cell source represents an important step toward generating universal platelets for transfusion as well as a potential strategy for the management of platelet refractoriness.

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Human erythropoiesis is a complex multistep process that involves the differentiation of early erythroid progenitors to mature erythrocytes. Here we show that it is feasible to differentiate and mature human embryonic stem cells (hESCs) into functional oxygen-carrying erythrocytes on a large scale (10 10 -10 11 cells/6-well plate hESCs). We also show for the first time that the oxygen equilibrium curves of the hESCderived cells are comparable with normal red blood cells and respond to changes in pH and 2,3-diphosphoglyerate. Although these cells mainly expressed fetal and embryonic globins, they also possessed the capacity to express the adult ␤-globin chain on further maturation in vitro. Polymerase chain reaction and globin chain specific immunofluorescent analysis showed that the cells increased expression of ␤-globin (from 0% to > 16%) after in vitro culture. Importantly, the cells underwent multiple maturation events, including a progressive decrease in size, increase in glycophorin A expression, and chromatin and nuclear condensation. This process resulted in extrusion of the pycnotic nuclei in up to more than 60% of the cells generating red blood cells with a diameter of approximately 6 to 8 m. The results show that it is feasible to differentiate and mature hESCs into functional oxygen-carrying erythrocytes on a large scale. (Blood. 2008;112:4475-4484) IntroductionHuman embryonic stem cells (hESCs) can be propagated and expanded in vitro indefinitely, providing a potentially inexhaustible and donorless source of cells for human therapy. Hematopoietic differentiation of hESCs has been extensively investigated in vitro, and hematopoietic precursors as well as differentiated progeny representing erythroid, myeloid, macrophage, megakaryocytic, and lymphoid lineages have been identified in differentiating hESC cultures. [1][2][3][4][5][6][7][8] Previous studies also generated primitive erythroid cells from hESCs by embryoid body formation and coculturing with stromal cells. [8][9][10] However, the efficient and controlled differentiation of hESCs into homogeneous red blood cell (RBC) populations with oxygen-carrying capacity has not been previously achieved.Mammalian erythropoiesis is a complex process that involves many steps, including the differentiation of early erythroid progenitors (burst-forming units-erythroid, BFU-E) via late erythroid progenitors (colony-forming units-erythroid, CFU-E), and finally morphologically recognizable erythroid precursors. 11 Nuclear condensation is a key event in the late stages of erythropoiesis, and enucleation is the final step in the development of mature erythrocytes, although the molecular and cellular mechanisms involved in these processes are poorly understood.Here we describe an efficient method to generate functional erythroid cells from hESCs under conditions suitable for scale-up. The cells possess oxygen-transporting capacity comparable with normal RBCs and respond to changes in pH and 2,3-diphosphoglycerate. We also show that they undergo a progressive decrea...

Human induced pluripotent stem cells (hiPSC) have been shown to differentiate into a variety of replacement cell types. Detailed evaluation and comparison with their human embryonic stem cell (hESC) counterparts is critical for assessment of their therapeutic potential. Using established methods, we demonstrate here that hiPSCs are capable of generating hemangioblasts/blast cells (BCs), endothelial cells, and hematopoietic cells with phenotypic and morphologic characteristics similar to those derived from hESCs, but with a dramatic decreased efficiency. Furthermore, in distinct contrast with the hESC derivatives, functional differences were observed in BCs derived from hiPSCs, including significantly increased apoptosis, severely limited growth and expansion capability, and a substantially decreased hematopoietic colony-forming capability. After further differentiation into erythroid cells, >1,000-fold difference in expansion capability was observed in hiPSC-BCs versus hESC-BCs. Although endothelial cells derived from hiPSCs were capable of taking up acetylated low-density lipoprotein and forming capillary-vascular-like structures on Matrigel, these cells also demonstrated early cellular senescence (most of the endothelial cells senesced after one passage). Similarly, retinal pigmented epithelium cells derived from hiPSCs began senescing in the first passage. Before clinical application, it will be necessary to determine the cause and extent of such abnormalities and whether they also occur in hiPSCs generated using different reprogramming methods. STEM CELLS 2010;28:704-712 Disclosure of potential conflicts of interest is found at the end of this article.