Name: competitive transplants to evaluate hematopoietic stem cell fitness
Uploaded: 2016-08-31T13:00:00
Description: Watch this Scientific Journal Video about Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness at JoVE.com

We are grateful to Roxann H&#233;tu-Arbour for assistance with the figure design and demonstration of the procedures. Research in the lab was supported by a Transition award from the Cole Foundation, Discovery grant no. 419226-2012 from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canada Foundation for Innovation (CFI Leaders Fund grant no. 31377). KMH is a Chercheur-Boursier Junior for the Fonds de recherche du Qu&#233;bec - Sant&#233; (FRQS).

All procedures described in this protocol have been approved by the institutional animal ethics committee and follow the Canadian Council on Animal Care guidelines.
Note: To maintain sterile/specific pathogen-free housing conditions, conduct all procedures involving direct handling of live mice inside a biological safety cabinet or a laminar flow hood. Clean or sterilize cages, restraining devices, housing materials, chow and water provided to the animals appropriately. Use only sterile, dispo...

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The protocol described here is designed to evaluate the relative fitness of donor (test) HSCs against known competitor HSCs. The situation of competition increases the relative sensitivity of the assay (more likely to detect moderate decreases in stem cell fitness) and provides an internal technical control for the efficacy of irradiation and injection. However, it should not be used as an absolute measure of HSC fitness; a decrease in competitive reconstitution does not automatically mean that the HSCs would not perform...

Hematopoiesis is a regenerative process that ensures the replenishing of blood cells that have been lost through injury, radiation and cell death. This process is ensured by hematopoietic stem cells (HSC) that largely reside in the adult bone marrow. In addition, hematopoietic stem cells can be used for therapeutic purposes in autoimmune disorders, hematological malignancies and immunodeficiencies1. There is thus a need to better understand the mechanisms that regulate HSC function, including their proliferative expansion and their ability to reach and engraft the recipient bone marrow after transplant. Although recent studies have reported several cell surface markers, including the SLAM family members CD150 and CD48, to prospectively enrich adult HSCs and fetal HSCs to approximately 50% purity2-4, the gold standard measure for functional HSCs remains an in vivo repopulating assay to determine their ability to re-establish blood cell production in an irradiated host5.
The in vivo clonal repopulating assay was initially developed by Till and McCulloch6 and has since been refined and expanded. As originally defined, HSCs ensure lifelong blood cell production through self-renewal and differentiation. The transfer of HSCs into an irradiated recipient thus allows us to assess: their ability to differentiate through the analysis of the different blood cell lineages (T lymphocytes, B lymphocytes, granulocytes, monocytes) and their capacity for self-renewal through serial transplantation. The assay would usually involve the comparison of the functionality and/or quantity of two populations of HSCs, e.g., cells coming from two mice of different genotypes or cells that have been treated or untreated with different factors that could influence the maintenance or expansion of HSCs in culture. Donor chimerism, or the contribution of transferred donor HSCs to blood cell production can then be determined by flow cytometry analysis in the peripheral blood and bone marrow using cell surface markers or other methods that will distinguish donor cells from the recipient, or host. The most widely used markers are certainly the two alleles for the gene Ptprc or CD45 leukocyte antigen7 that we have chosen for the examples provided below.
The clonal repopulation assay can be either competitive or non-competitive. In a non-competitive setting, control and test HSCs are transferred into separate recipient mice and the outcome for each cell type will be independent of the other. In a competitive setting, the function of both test and control HSCs is measured against a population of competitor HSCs. The protocol described here uses the competitive setting but can also be adapted for non-competitive situations. Both approaches have their advantages and limitations, and we will compare them in detail in the discussion. We also describe different approaches to ensure equity in the number of transplanted HSCs, explain how to adapt the assay for the quantification of HSCs by limiting dilution assay (LDA), and provide examples of both successful and unsuccessful transplants for the interpretation of results.

<table border="0" cellpadding="0" cellspacing="0" width="703">
	<tbody>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Microtainer tubes with K2EDTA</td>
			<td style="width:89px;">BD Biosciences</td>
			<td style="width:112px;"></td>
			<td style="width:119px;">365974</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">20G needle</td>
			<td style="width:89px;">BD Syringe</td>
			<td style="width:112px;"></td>
			<td style="width:119px;"></td>
			<td>For blood sampling from the mandibular vein</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LabQuake Shaker rotisserie</td>
			<td style="width:89px;">Thermo&#160; Scientific</td>
			<td style="width:112px;"></td>
			<td style="width:119px;">C415110</td>
			<td>Any other rotating mixer will work as well to prevent coagulation of blood samples</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Purified anti-mouse CD16/CD32 (clone 2.4G2, Fc Block)</td>
			<td style="width:89px;">BD Biosciences</td>
			<td style="width:112px;">2.50</td>
			<td style="width:119px;">553142</td>
			<td>Alternatively use clone 93 from eBioscience (cat # 14-0161) or Biolegend (cat# 101310)&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pe-Cy7-conjugated anti-mouse CD3e (clone 145-2C11)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">0.25</td>
			<td style="width:119px;">25-0031</td>
			<td>For most flow cytometry antibodies, the clone is important but the colours and companies can vary depending on the available equipment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PE-conjugated anti-mouse CD19 (clone 1D3)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">0.25</td>
			<td style="width:119px;">12-0193</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">APC-eFluor780 (APC-Cy7 equivalent)-conjugated anti-mouse GR1 (clone RB6-8C5)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">0.25</td>
			<td style="width:119px;">47-5931</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FITC-conjugate anti-mouse CD45.1 (clone A20)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">2.50</td>
			<td style="width:119px;">11-0453</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">eFluor450-conjugated anti-mouse CD45.2 (clone 104)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">1.00</td>
			<td style="width:119px;">48-0454</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Biotinylated anti-human/mouse CD45R (B220) (clone RA3-6B2)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">1.25</td>
			<td style="width:119px;">13-0452</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biotinylated anti-mouse CD3e (clone 145-2C11)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">1.25</td>
			<td style="width:119px;">13-0031</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biotinylated anti-mouse CD11b (clone M1/70)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">1.25</td>
			<td style="width:119px;">13-0112</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biotinylated anti-mouse GR1 (clone RB6-8C5)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">1.25</td>
			<td style="width:119px;">13-5931</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biotinylated anti-mouse TER119 (clone TER119)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">0.63</td>
			<td style="width:119px;">13-5921</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">V500 streptavidin</td>
			<td style="width:89px;">BD Biosciences</td>
			<td style="width:112px;">0.50</td>
			<td style="width:119px;">561419</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PE-conjugated anti-mouse CD117 (clone 2B8)</td>
			<td style="width:89px;">BD Biosciences</td>
			<td style="width:112px;">0.25</td>
			<td style="width:119px;">553355</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PE-Cy7-conjugated anti-mouse Ly6A/E (Sca1) (clone D7)</td>
			<td style="width:89px;">BD Biosciences</td>
			<td style="width:112px;">0.25</td>
			<td style="width:119px;">558162</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">PerCP-eFluor710-conjugated anti-mouse CD135 (clone A2F10)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">0.50</td>
			<td style="width:119px;">46-1351</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Alexa fluor 647-conjugated anti-mouse CD150 (clone TC15-12F12.2)</td>
			<td style="width:89px;">Biolegend</td>
			<td style="width:112px;">0.63</td>
			<td style="width:119px;">115918</td>
			<td>BD Biosciences and eBioscience do not carry the same clone</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1ml tuberculin syringe with 27G needle</td>
			<td style="width:89px;">BD Syringe</td>
			<td style="width:112px;"></td>
			<td style="width:119px;">309623</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1ml tuberculin syringe with 25G needle</td>
			<td style="width:89px;">BD Syringe</td>
			<td style="width:112px;"></td>
			<td style="width:119px;">309626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">70 um cell strainer</td>
			<td style="width:89px;">BD Falcon</td>
			<td style="width:112px;"></td>
			<td style="width:119px;">352350</td>
			<td></td>
		</tr>
	</tbody>
</table>

competitive transplants to evaluate hematopoietic stem cell fitness

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to reestablish blood cell production for the lifespan of the recipient. This protocol explains how to set up a functional assay to compare the HSC capacities of two different populations of cells, such as bone marrow from mice of two different genotypes, and how to analyze the recipient mice by flow cytometry. The protocol uses HSC equivalents rather than cell sorting for standardization and discusses the advantages and disadvantages of both approaches. We further discuss different variations to the basic protocol, including serial transplants, limiting dilution assays, homing assays and non-competitive transplants, including the advantages and preferred uses of these varied approaches. These assays are central for the study of HSC function and could be used not only for the investigation of fundamental HSC intrinsic aspects of biology but also for the development of preclinical assays for bone marrow transplant and HSC expansion in culture.

A general description of the competitive transplant setting, including secondary transplants (discussed further below) can be found in Figure 1. A representative analysis for pre-transplant bone marrow HSCs can be found in Figure 2. More detailed information on the exclusion of doublets and dead cells can be found elsewhere9.
Figures 3 and 4 provide e...

Watch this Scientific Journal Video about Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness at JoVE.com

Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness

This protocol provides step-by-step guidelines for setting up competitive mouse bone marrow transplant experiments to study hematopoietic stem/progenitor cell function without prior purification of stem cells by cell sorting.

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to ...

The overall goal of this experiment is to determine the fitness of bone marrow hematopoietic stem cells in vivo using a competitive transplant assay. So this method can help answer key questions in the field of stem cell biology and hematopoietic cell transplantation about the impact of various manipulations on stem cell self-renewal after transplant. The main advantages of this technique are that it requires a limited number of animals and that it does not involve cell sorting.Though this method can provide insight into stem cell function, it can be also useful in other fields such as immunology, through the use of mixed bone marrow chimeras. The initial development of this technique in the 1960s paved the way for researchers in the field of stem cell biology to explore the identity and function of hematopoietic stem cells in humans and mice. Demonstrating the procedure will be Roxanne Hetu-Arbour, a Master's student in the lab, and Edward Owusu Kwarteng, a PhD student.Begin by placing the mouse inside a biological safety cabinet on a surgical platform and wet the skin and fur with 70%ethanol. Next, take hold of a hind foot and cut the skin up along the leg. Proceed to pull away the skin and cut away the excess muscle.Then, with the scissor blades against the pelvic bones as a counter-force, pull on the leg bone to dislocate the femur. Detach the tibia and the femur from the kneecap, then remove the foot, and remove the remaining bits of muscle and tendons. Then, place the bones in two to three milliliters of sterile PBS in one well of a six-well tissue culture plate.When all of the bones have been harvested, grasp the bones one at a time with a forceps, and insert a 25-gauge needle attached to a one-milliliter syringe filled with sterile PBS into one end of the bone. Depress the plunger, collecting the cells in a 15-milliliter conical tube until the center of each bone is white. Using a 22-gauge needle, dissociate the cells into a single cell suspension by repeatedly passing them through the needle tip.After all of the bone marrow has been collected, filter the cells through a 70-micron nylon strainer and count the resulting cell suspensions. Then, centrifuge the bone marrow cells, remove the supernatant, and dilute the samples to a one times ten to the eighth cells per milliliter concentration in fresh PBS, on ice. To establish donor hematopoietic stem cell equivalents, transfer 30 microliter aliquots of cells into five-milliliter round-bottomed polystyrene tubes and add an equal volume of unlabeled antibody against CD16 and CD32 to the samples for five minutes at room temperature to block any non-specific staining.Next, incubate the cells in 90 microliters of the appropriate fluorochrome conjugated primary antibody master mix on ice, protected from light. After 30 minutes, add two milliliters of staining buffer to the cells and vortex the tubes. Follow this with centrifugation.Decant the supernatants and resuspend the pellets by flicking. Then, label the cells with 10 microliters of fluorochrome conjugated streptavidin on ice for 20 minutes in the dark. At the end of the incubation, wash the cells in two more milliliters of buffer and analyze the samples by flow cytometry.Using one sample as a baseline, establish the estimated hematopoietic stem cell equivalents for each of the samples. After calculating the number of hematopoietic stem cells per sample as outlined in the text, mix the competitor and test hematopoietic stem cells at a one-to-one ratio and adjust the final volume of the cells to 200 microliters per injection in sterile PBS. For adoptive transfer of the hematopoietic stem cells, warm up an irradiated recipient mouse with a heat lamp.Then aspirate 750 microliters of cells into one one-milliliter tuberculin syringe equipped with a 27-gauge needle and remove the bubbles. Next, with the mouse in the appropriate restraining device, wipe the injection site with ethanol. Insert the needle into a lateral tail vein, bevel side up and parallel to the skin, and gently depress the plunger.When all of the cells have been injected, remove the needle and compress the injection site with sterile gauze for a few seconds to stop the bleeding. Then place the mouse in a clean cage. To analyze the peripheral blood, pierce the facial skin near the hairless spot located under the jawbone and collect one to two drops of blood in an EDTA tube.Place the tube in a rotator to prevent clotting while waiting to proceed. Next, mix one milliliter of freshly-prepared, room temperature, red blood cell lysis buffer with the blood and transfer the sample to a five-milliliter, round-bottomed polystyrene tube. After four minutes at room temperature, add four volumes of ice-cold PBS to each tube and cover the opening with a cap.Mix the tube a few times by inversion and immediately place the cells on ice. After centrifugation, decant the clear red supernatants and resuspend the pellets by flicking. Then add two milliliters of staining buffer to the cells and briefly vortex them.Follow this with a a second centrifugation. Resuspend the pellets by flicking and proceed with flow cytometry staining as detailed in the text. The competitor-derived cells should be present in all three of the myeloid, B-lymphoid, and T-lymphoid lineages.The competitor and donor cells are also found within both lineage negative and hematopoietic stem cell bone marrow cell populations. When the donor hematopoietic stem cells are functionally equivalent to the competitor cells, the proportions of the donor and the competitor-derived cells are equal. The residual host cells are typically almost exclusively T lymphocytes, as these cells are more radioresistant.When the donor cells are observed at a much lower proportion of peripheral blood leukocytes than the competitor cells, some aspects of hematopoietic stem cell function are defective, and warrant further investigation. When the donor and competitor cells are both present in low numbers and the host cells represent the majority of peripheral blood cells, the transplant is considered unsuccessful, and no conclusions can be drawn about the relative functionality of the donor versus the competitor cells. Once mastered, the bone marrow preparation and the transplantation steps can be completed in six to seven hours if they are performed properly.While attempting this procedure, it is important to remember to maintain sterile conditions during the cell preparation and the animal manipulation steps. Following this procedure, other methods, like a limiting dilution assay, can be performed to answer additional questions regarding the functional stem cell numbers. After watching this video, you should have a good understanding of how to perform competitive bone marrow transplants.Don't forget that working with irradiation can be extremely hazardous, and that the appropriate precautions should always be taken while performing this procedure.

competitive-transplants-to-evaluate-hematopoietic-stem-cell-fitness

Research

JoVE Journal

Developmental Biology

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

Automated Quantification of Hematopoietic Cell &amp;#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

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.

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 ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Detection of Residual Donor Erythroid Progenitor Cells after Hematopoietic Stem Cell Transplantation for Patients with Hemoglobinopathies

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell disease. This observation has tremendous implications, as subsequent therapeutic immunomodulation strategies can improve clinical outcome. Conventionally, polymerase chain reaction-based analysis of short tandem repeats is used to identify chimerism in donor-derived blood cells. However, this method is restricted to nucleated cells and cannot distinguish between dissociated single-cell lineages. We applied the analysis of short tandem repeats to flow cytometric-sorted hematopoietic progenitor cells and compared this with the analysis of short tandem repeats obtained from selected burst-forming unit - erythroid colonies, both collected from the bone marrow. With this method we are able to demonstrate the different proliferation and differentiation of donor cells in the erythroid compartment. This technique is eligible to complete current monitoring of chimerism in the stem cell transplant setting and thus may be applied in future clinical studies, stem cell research and design of gene therapy trials.

Quantification of donor-derived cells is required to monitor engraftment after stem cell transplantation in patients with hemoglobinopathies. A combination of flow cytometry-based cell sorting, colony formation assay, and subsequent analysis of short tandem repeats may be used to assess the proliferation and differentiation of progenitors in the erythroid compartment.

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell ...

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell lineages that comprise mature blood. Isolation and identification of these HSPCs is difficult because they are defined ex post facto; they can only be defined after their differentiation into specific cell lineages. Over the past few decades, the zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop ex utero, and by 48 h post-fertilization (hpf) have generated definitive HSPCs. Assays to assess HSPC differentiation and proliferation capabilities have been developed, utilizing transplantation and subsequent reconstitution of the hematopoietic system in addition to visualizing specialized transgenic lines with confocal microscopy. However, these assays are cost prohibitive, technically difficult, and time consuming for many laboratories. Development of an in vitro model to assess HSPCs would be cost effective, quicker, and present fewer difficulties compared to previously described methods, allowing laboratories to quickly assess mutagenesis and drug screens that affect HSPC biology. This novel in vitro assay to assess HSPCs is performed by plating dissociated whole zebrafish embryos and adding exogenous factors that promote only HSPC differentiation and proliferation. Embryos are dissociated into single cells and plated with HSPC-supportive colony stimulating factors that cause them to generate colony forming units (CFUs) that arise from a single progenitor cell. These assays should allow more careful examination of the molecular pathways responsible for HSPC proliferation, differentiation, and regulation, which will allow researchers to understand the underpinnings of vertebrate hematopoiesis and its dysregulation during disease.

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Here, we present a simple method to quantitate hematopoietic stem and progenitor cells (HSPCs) in embryonic zebrafish. HSPCs from dissociated zebrafish are plated in methylcellulose with supportive factors, differentiating into mature blood. This allows the detection of blood defects and allows drug screening to be easily conducted.

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early embryo and the lack of assays that functionally identify the long-term multilineage engraftment potential of individual putative HSC precursors. Here, we describe methodology that enables the isolation and characterization of functionally validated HSC precursors at the single cell level. First, we utilize index sorting to catalog the precise phenotypic parameter of each individually sorted cell, using a combination of phenotypic markers to enrich for HSC precursors with additional markers for experimental analysis. Second, each index-sorted cell is co-cultured with vascular niche stroma from the aorta-gonad-mesonephros (AGM) region, which supports the maturation of non-engrafting HSC precursors to functional HSC with multilineage, long-term engraftment potential in transplantation assays. This methodology enables correlation of phenotypic properties of clonal hemogenic precursors with their functional engraftment potential or other properties such as transcriptional profile, providing a means for the detailed analysis of HSC precursor development at the single cell level.

Here we present methodology for the clonal analysis of hematopoietic stem cell precursors during murine embryonic development. We combine index sorting of single cells from the embryonic aorta-gonad-mesonephros region with endothelial cell co-culture and transplantation to characterize the phenotypic properties and engraftment potential of single hematopoietic precursors.

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early ...

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute leukemia. NUP98-HOXD13 (NHD13) transgenic mice recapitulate human MDS in terms of peripheral blood cytopenias, dysplasia, and transformation to acute leukemia. We previously demonstrated that MDS could be transferred from a genetically engineered mouse with MDS to wild-type recipients by transplanting MDS bone marrow nucleated cells (BMNC). To more clearly understand the MDS cell of origin, we have developed approaches to transplant specific, immunophenotypically defined hematopoietic subsets. In this article, we describe the process of isolating and transplanting specific populations of hematopoietic stem and progenitor cells. Following transplantation, we describe approaches to assess the efficiency of transplantation and persistence of the donor MDS cells.

We describe the use of hematopoietic stem cell transplantation (HSCT) to assess the malignant potential of genetically engineered hematopoietic cells. HSCT is useful for evaluating various malignant hematopoietic cells in vivo as well as generating a large cohort of mice with myelodysplastic syndromes (MDS) or leukemia to evaluate novel therapies.

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood ...