This work has been supported by the Department of Internal Medicine, Division of Hematology, Washington University School of Medicine. CD was supported by T32HL007088 from the National Heart, Lung, and Blood Institute. CMS was supported by an American Society of Hematology Scholar Award.

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The authors declare that they have no competing financial interests.

This protocol describes a rapid, serum-free, stroma-free method for the differentiation of either primitive or definitive hematopoietic progenitors. Mesodermal specification of either primitive or definitive hematopoietic progenitors can be reliably achieved using our protocol, which uniquely exploits small molecule inhibitors of canonical WNT signaling. Stage-specific WNT activation by the GSK3&#946; inhibitor CHIR9902133 gives rise to definitive hematopoietic mesoderm, whereas WNT inhibition by ...

Human pluripotent stem cells (hPSCs) are defined as encompassing both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), and have the unique capability of not only undergoing self-renewal under appropriate growth conditions, but also, the capacity to differentiate into all cell types derived from the three germ layers: endoderm, mesoderm, and ectoderm1. Due to these unique abilities, hPSCs hold great promise for regenerative medicine, disease modeling, and cell-based therapies2. While multiple cell types have been successfully differentiated from hPSCs, one significant challenge is the in vitro specification of exclusively adult-like hPSC-derived hematopoietic stem cells (HSCs) and definitive hematopoietic progenitors.
One likely barrier to the development of human HSCs from hPSCs is the presence of multiple hematopoietic programs within the human embryo3. The first program which emerges, termed &#34;primitive hematopoiesis,&#34;&#160;originates within the extraembryonic yolk sac tissue and is best characterized by its transient production of erythroblast progenitors (EryP-CFC), macrophages, and megakaryocytes. Notably, this program does not give rise to HSCs, nor does it give rise to T and B lymphoid progenitors. However, the yolk sac does transiently give rise to restricted definitive hematopoietic progenitors, such as the erythro-myeloid progenitor (EMP4,5,6,7,8) and the erythroid-deficient lymphoid-primed multipotent progenitor (LMPP9). However, neither EMPs nor LMPPs are fully multipotent, or capable of HSC-like engraftment in adult recipients. In contrast, later in development, the classically defined &#34;definitive&#34; hematopoietic program is specified in the aorta-gonad-mesonephros region of the embryo proper, giving rise to all adult hematopoietic lineages, including the HSC. The specification of these intra-embryonic definitive hematopoietic cells occurs in a Notch-dependent fashion, via an endothelial-to-hematopoietic transition from hemogenic endothelium (HE)3,10,11,12,13,14. Aside from reconstitution capacity, the multilineage potential and Notch-dependence of these cells can be used to distinguish these definitive hematopoietic progenitors from the EMP and the LMPP (reviewed in references3,13).
Understanding the mechanism(s) governing primitive and definitive hematopoietic specification from hPSCs is likely critical to the reproducible production of definitive hematopoietic progenitors across a variety of hPSC lines. Until recently, hPSC differentiation protocols that could separate multipotent primitive and definitive hematopoietic progenitors did not exist15,16,17,18,19,20,21,22,23,24,25. Many approaches using fetal bovine serum (FBS) and/or stromal co-culture first outlined the hematopoietic potential of hPSC differentiation, with mixtures of primitive and definitive hematopoietic potential15,16,17,19,22,23,25. Further, many serum-free hematopoietic protocols have described the signal requirements for the specification of mesoderm from hPSCs that harbors hematopoietic potential18,20,21,24. However, as these methods still gave rise to heterogeneous mixtures of both programs, their use in clinical applications and understanding developmental mechanisms may be limited.
We have recently built on these studies, having outlined the stage-specific signal requirements for ACTIVIN/NODAL and WNT signaling in primitive and definitive hematopoietic specification from hPSC-derived mesoderm18,26. The latter was particularly unique, as its use of stage-specific WNT signal manipulation allows for the specification of either exclusively primitive or exclusively definitive hematopoietic progenitors26. During mesoderm specification, the inhibition of canonical WNT signaling with the PORCN inhibitor IWP2 results in the specification of CD43+ EryP-CFC and myeloid progenitors, with no detectable lymphoid potential. In sharp contrast, stimulation of canonical WNT signaling with the GSK3&#946; inhibitor, CHIR99021, during the same stage of differentiation resulted in the complete absence of detectable CD43+ EryP-CFC, while simultaneously leading to the specification of CD34+CD43&#8722; HE. This population possessed myeloid, HBG-expressing erythroid, and T-lymphoid potential. Subsequent analyses identified this HE as lacking the expression of CD7327,28 and CD18428, and its hematopoietic potential was NOTCH-dependent28. Further, single-cell clonal analyses demonstrated that these definitive hematopoietic lineages could be derived from multipotent single cells28. Taken together, these studies indicate that stage-specific WNT signaling manipulation can specify either pure primitive hematopoietic progenitors, or multipotent NOTCH-dependent definitive hematopoietic progenitors.
Here, we outline our differentiation strategy that yields exclusively primitive or definitive hematopoietic progenitors, via manipulation of canonical WNT signaling during mesodermal patterning, and their downstream hematopoietic lineage assays. This protocol is of great value to investigators who are interested in the production of either primitive or definitive hematopoietic progenitors from hPSCs for regenerative medicine applications.

<table><tbody><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Medium (IMD)</td><td>Corning</td><td>10-016</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS), ES cell rated</td><td>Gemini Bioproducts</td><td>100-500</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Hyclone</td><td>SH30396.03</td><td></td></tr><tr><td>L-glutamine, 200 mM solution</td><td>Life Technologies</td><td>25030-081</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Life Technologies</td><td>15070-063</td><td></td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Life Technologies</td><td>25200056</td><td></td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Life Technologies</td><td>25300054</td><td></td></tr><tr><td>Gelatin, porcine skin, Type A</td><td>Sigma-Aldrich</td><td>G1890</td><td></td></tr><tr><td>Alpha-MEM</td><td>Life Technologies</td><td>12000-022</td><td></td></tr><tr><td>DMEM-F12</td><td>Corning</td><td>10-092-CV</td><td></td></tr><tr><td>Knock-out serum replacement</td><td>Life Technologies</td><td>10828028</td><td>&quot;KOSR&quot;</td></tr><tr><td>Non-essential amino acids (NEAA)</td><td>Life Technologies</td><td>11140050</td><td></td></tr><tr><td>b-mercaptoethanol, 55 mM solution</td><td>Life Technologies</td><td>21985023</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Sigma-Aldrich</td><td>H1758</td><td></td></tr><tr><td>Fraction V, Bovine Serum Albumin</td><td>Fisher Scientific</td><td>BP1605</td><td></td></tr><tr><td>Ham&#039;s F12</td><td>Corning</td><td>10-080</td><td></td></tr><tr><td>N2 supplement</td><td>Life Technologies</td><td>17502048</td><td></td></tr><tr><td>B27 supplement, no vitamin A</td><td>Life Technologies</td><td>12587010</td><td></td></tr><tr><td>Stempro-34 (SP34)</td><td>Life Technologies</td><td>10639011</td><td>&quot;SP34&quot;</td></tr><tr><td>Growth factor reduced Matrigel</td><td>Corning</td><td>354230</td><td>&quot;MAT&quot;</td></tr><tr><td>L-absorbic acid</td><td>Sigma-Aldrich</td><td>A4403</td><td></td></tr><tr><td>Human serum transferrin</td><td>Sigma-Aldrich</td><td>10652202001</td><td></td></tr><tr><td>Monothioglycerol (MTG)</td><td>Sigma-Aldrich</td><td>M6145</td><td></td></tr><tr><td>Collagenase B</td><td>Roche</td><td>11088831001</td><td></td></tr><tr><td>Collagenase II</td><td>Life Technologies</td><td>17101015</td><td></td></tr><tr><td>DNaseI</td><td>Calbiochem</td><td>260913</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Life Technologies</td><td>14190144</td><td></td></tr><tr><td>bFGF</td><td>R&amp;amp;D Systems</td><td>233-FB</td><td></td></tr><tr><td>BMP4</td><td>R&amp;amp;D Systems</td><td>314-BP</td><td></td></tr><tr><td>Activin A</td><td>R&amp;amp;D Systems</td><td>338-AC</td><td></td></tr><tr><td>VEGF</td><td>R&amp;amp;D Systems</td><td>293-VE</td><td></td></tr><tr><td>SCF</td><td>R&amp;amp;D Systems</td><td>255-SC</td><td></td></tr><tr><td>IGF-1</td><td>R&amp;amp;D Systems</td><td>291-G1</td><td></td></tr><tr><td>IL-3</td><td>R&amp;amp;D Systems</td><td>203-IL</td><td></td></tr><tr><td>IL-6</td><td>R&amp;amp;D Systems</td><td>206-IL</td><td></td></tr><tr><td>IL-7</td><td>R&amp;amp;D Systems</td><td>207-IL</td><td></td></tr><tr><td>IL-11</td><td>R&amp;amp;D Systems</td><td>218-1L</td><td></td></tr><tr><td>TPO</td><td>R&amp;amp;D Systems</td><td>288-TP</td><td></td></tr><tr><td>EPO</td><td>Peprotech</td><td>100-64</td><td></td></tr><tr><td>Flt-3 ligand (FLT3-L)</td><td>R&amp;amp;D Systems</td><td>308-FK</td><td></td></tr><tr><td>CHIR99021</td><td>Tocris</td><td>4423</td><td></td></tr><tr><td>IWP2</td><td>Tocris</td><td>3533</td><td></td></tr><tr><td>Angiotensin II</td><td>Sigma-Aldrich</td><td>A9525</td><td></td></tr><tr><td>Losartan Potassium</td><td>Tocris</td><td>3798</td><td></td></tr><tr><td>CD4 PerCP Cy5.5 Clone RPA-T4</td><td>BD Biosciences</td><td>560650</td><td>Dilution 1:100; T cell assay</td></tr><tr><td>CD8 PE Clone RPA-T8</td><td>BD Biosciences</td><td>561950</td><td>Dilution 1:10; T cell assay</td></tr><tr><td>CD34 APC Clone 8G12</td><td>BD Biosciences</td><td>340441</td><td>Dilution 1:100; EHT assay</td></tr><tr><td>CD34 PE-Cy7 Clone 8G12</td><td>BD Biosciences</td><td>348801</td><td>Dilution 1:100; Hemogenic endothelium</td></tr><tr><td>CD43 FITC Clone 1G10</td><td>BD Biosciences</td><td>555475</td><td>Dilution 1:10; Hemogenic endothelium</td></tr><tr><td>CD45 APC-Cy7 Clone 2D1</td><td>BD Biosciences</td><td>557833</td><td>Dilution 1:50; T cell assay</td></tr><tr><td>CD45 eFluor450 Clone 2D1</td><td>BD Biosciences</td><td>642284</td><td>Dilution 1:50; EHT assay</td></tr><tr><td>CD56 APC Clone B159</td><td>BD Biosciences</td><td>555518</td><td>Dilution 1:20; T cell assay</td></tr><tr><td>CD73 PE Clone AD2</td><td>BD Biosciences</td><td>550257</td><td>Dilution 1:50; Hemogenic endothelium</td></tr><tr><td>CD184 APC Clone 12G5</td><td>BD Biosciences</td><td>555976</td><td>Dilution 1:50; Hemogenic endothelium</td></tr><tr><td>4&#039;,6-diamidino-2-phenylindole (DAPI)</td><td>BD Biosciences</td><td>564907</td><td>Dilution 1:10,000; T cell assay</td></tr><tr><td>OP9 DL4 cells</td><td></td><td></td><td>Holmes, R. and J.C. Zuniga-Pflucker. Cold Spring Harb Protoc, 2009. 2009(2): p. pdb prot5156</td></tr><tr><td>MethoCult H4034</td><td>Stemcell Technologies</td><td>4034</td><td>&quot;MeC&quot;</td></tr><tr><td>Milli-Q water purification system</td><td>EMD Millipore</td><td></td><td></td></tr><tr><td>5% CO2 incubator</td><td></td><td></td><td>Set at 37 C</td></tr><tr><td>Multigas incubator</td><td></td><td></td><td>Set at 37 C, 5% CO2, 5% O2</td></tr><tr><td>6 well tissue culture plate</td><td>Corning</td><td>353046</td><td></td></tr><tr><td>24 well tissue culture plate</td><td>Corning</td><td>353226</td><td></td></tr><tr><td>6 well low-adherence tissue culture plate</td><td>Corning</td><td>3471</td><td></td></tr><tr><td>24 well low-adherence tissue culture plate</td><td>Corning</td><td>3473</td><td></td></tr><tr><td>35 mm tissue culture dishes</td><td>Corning</td><td>353001</td><td></td></tr><tr><td>Blunt-end needle, 16 gauge</td><td>Corning</td><td>305198</td><td></td></tr><tr><td>3 cc syringes</td><td>Corning</td><td>309657</td><td></td></tr><tr><td>5 mL polypropylene test tube</td><td>Corning</td><td>352063</td><td></td></tr><tr><td>5 mL polystyrene test tube</td><td>Corning</td><td>352058</td><td></td></tr><tr><td>15 mL polypropylene conical</td><td>Corning</td><td>430791</td><td></td></tr><tr><td>50 mL polypropylene conical</td><td>Corning</td><td>430921</td><td></td></tr><tr><td>2 mL serological pipette</td><td>Corning</td><td>357507</td><td></td></tr><tr><td>5 mL serological pipette</td><td>Corning</td><td>4487</td><td></td></tr><tr><td>10 mL serological pipette</td><td>Corning</td><td>4488</td><td></td></tr><tr><td>25 mL serological pipette</td><td>Corning</td><td>4489</td><td></td></tr><tr><td>Cell scrapers</td><td>Corning</td><td>353085</td><td></td></tr><tr><td>2.0 mL cryovials</td><td>Corning</td><td>430488</td><td></td></tr><tr><td>5 mL test tube with 40 &amp;micro;M cell strainer</td><td>Corning</td><td>352235</td><td></td></tr><tr><td>40 &amp;micro;M cell strainer</td><td>Corning</td><td>352340</td><td></td></tr><tr><td>Cell culture centrifuge</td><td></td><td></td><td></td></tr><tr><td>Biosafety hood</td><td></td><td></td><td></td></tr><tr><td>FACS AriaII or equivalent</td><td></td><td></td><td></td></tr><tr><td>LSRii or equivalent</td><td></td><td></td><td></td></tr><tr><td>FlowJo software</td><td>TreeStar</td><td></td><td></td></tr><tr><td>Water bath</td><td></td><td></td><td>Set at 37 C</td></tr><tr><td>0.22 &amp;micro;M filtration system</td><td>Corning</td><td></td><td></td></tr><tr><td>Autoclave</td><td></td><td></td><td></td></tr><tr><td>4 C refrigerator</td><td></td><td></td><td></td></tr><tr><td>-20 C Freezer</td><td></td><td></td><td></td></tr><tr><td>-80 C Freezer</td><td></td><td></td><td></td></tr></tbody></table>

directed differentiation of primitive and definitive hematopoietic progenitors from human pluripotent stem cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

A schematic depicting the induction of primitive and definitive hematopoietic progenitors from hPSCs is illustrated in Figure 1. Mesoderm patterning by canonical WNT signaling occurs during days 2 - 3 of differentiation, followed by hematopoietic progenitor specification.
Representative flow cytometric analysis and colony forming methylcellulose assays of hPSC-derived hematopoietic differentiation c...

Watch this Scientific Journal Video about Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells at JoVE.com

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

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Research

JoVE Journal

Developmental Biology

11.0K Views.  Washington University in St. Louis. Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

Video: Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro is essential for mechanistic studies of the endothelial-hematopoietic transition and hematopoietic specification. Here, we describe a method for the efficient induction of HE from human pluripotent stem cells (hPSCs) by way of overexpression of different sets of transcription factors. The combination of ETV2 and GATA1 or GATA2 TFs is used to induce HE with pan-myeloid potential, while a combination of GATA2 and TAL1 transcription factors allows for the production of HE with erythroid and megakaryocytic potential. The addition of LMO2 to GATA2 and TAL1 combination substantially accelerates differentiation and increases erythroid and megakaryocytic cells production. This method provides an efficient and rapid means of HE induction from hPSCs and allows for the observation of the endothelial-hematopoietic transition in a culture dish. The protocol includes hPSCs transduction procedures and post-transduction analysis of HE and blood progenitors.

This protocol describes the efficient induction of hemogenic endothelium and multipotential hematopoietic progenitors from human pluripotent stem cells via the forced expression of transcription factors.

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro ...

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

Immunology and Infection

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine induced differentiation of CD34+ hematopoietic stem and progenitor cells to the four myeloid lineage cells. CD34+ cells are isolated from human umbilical cord blood and co-cultured with MS-5 stromal cells in the presence of cytokines. Immunophenotypic characterization of the stem and progenitor cells, and the differentiated myeloid lineage cells are described. Using this protocol, CD34+ cells may be incubated with small molecules or transduced with lentiviruses to express myeloid disease mutations to investigate their impact on myeloid differentiation.

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Here, we present a protocol for immunophenotypic characterization and cytokine induced differentiation of cord blood derived CD34+ hematopoietic stem and progenitor cells to the four myeloid lineages. The applications of this protocol include investigations on the effect of myeloid disease mutations or small molecules on myeloid differentiation of the CD34+ cells.

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine ...

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

Neuroscience

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3&#946; inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...

Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a single-cell suspension for further use in embryoid body induction. Following mesodermal induction, embryoid bodies undergo hematopoietic differentiation, producing a floating hematopoietic cell population. Subsequently, the harvested hematopoietic cells undergo a macrophage colony-stimulating factor maturation step and, finally, osteoclast differentiation. After osteoclast differentiation, osteoclasts are characterized by staining for TRAP in conjunction with a methyl green nuclear stain. Osteoclasts are observed as multinucleated, TRAP+ polykaryons. Their identification can be further supported by Cathepsin K staining. Bone and mineral resorption assays allow for functional characterization, confirming the identity of bona fide osteoclasts. This protocol demonstrates a robust and versatile method to differentiate human osteoclasts from iPSCs and allows for easy adoption in applications requiring large quantities of functional human osteoclasts. Applications in the areas of bone research, cancer research, tissue engineering, and endoprosthesis research could be envisioned.

This protocol presents the differentiation of human osteoclasts from induced pluripotent stem cells (iPSCs) and describes methods for the characterization of osteoclasts and osteoclast precursors.

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a ...

Hepatic Progenitor Specification from Pluripotent Stem Cells using a Defined Differentiation System

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to shortages in donor organ availability. Organ scarcity also affects the routine supply of human hepatocytes for basic research and the clinic. Therefore, the development of renewable sources of human liver progenitor cells is desirable and is the goal of this study. To be able to effectively generate and deploy human liver progenitors on a large scale, a reproducible hepatic progenitor differentiation system was developed. This protocol aids experimental reproducibility between users in a range of cell cultureware formats and permits differentiations using both, human embryonic and induced pluripotent stem cell lines. These are important advantages over current differentiation systems that will enhance the basic research and may pave the way towards clinical product development.

The goal of this article is to provide a standardized approach to induce human hepatic progenitor differentiation from pluripotent stem cells. The development of this procedure with ready-to-use media formulations offer the user a facile system to generate human liver cells for biomedical research and translation.

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to ...