We would like to thank David R. Rodriguez, M.A. for the graphic design and photography in this manuscript. This work was supported by funding from the GCCRI, the NIH/NIA (5R21AG033339) and the Cancer Center Support Grant (P30CA054174) to the UTHSCSA Flow Cytometry Core facility and the UTHSCSA Advanced Nucleic Acids Core Facility.

The authors declare&#160;no competing financial interests.

The in vivo mutagenesis assay described herein is based on the LacI transgenic mouse model originally generated by Kohler et al.18 This model utilizes a &#955; phage vector carrying a lacI reporter gene. The two cos sites flanking the vector allow for a relatively simple recovery and subsequent packaging into infectious phage particles, used to infect E. coli. A blue plaque will be generated by phage-infected E. coli that contain a mutated LacI...

In most tissues, differentiated cells have a limited life-span. To maintain functional integrity, long-lived, tissue-specific stem cells continuously produce progenitor cells that in turn give rise to the fully differentiated cells required for the function of that particular tissue. Stem cells also replenish their own compartment through a process called self-renewal. Thus, stem cells are responsible for maintaining the functional integrity of the tissue they reside in. Therefore, it is imperative that they are equipped with robust mechanisms to sense and potentially repair damaged DNA. If not, they may acquire multiple genomic (potentially harmful) perturbations, which can be inherited by their progeny. Understanding how stem cells safe-guard their genome during the life span of an organism is an important question and may help us understand why genomic instability is linked with cancer and some other age-related diseases (reviewed in1,2).
Controlling genomic integrity of a tissue at the level of stem cells or early progenitor cell populations can be achieved by either eliminating defective stem (or progenitor) cells via cell death, senescence or differentiation, and/or by efficient repair of damaged DNA. Recent studies have demonstrated that it is possible to measure certain types of DNA repair directly in these rare populations3-6. It was found that, for example in the hematopoietic system, double strand DNA breaks can be repaired by homologous recombination (HR) or non-homologous end joining (NHEJ), the latter being a repair process of lower fidelity and thus increased risk of making errors. Both are being utilized in hematopoietic stem cells (HSCs)4,5, however, in mice it seems it is predominantly NHEJ in HSCs whereas early progenitors cells utilize HR4. A similar observation was made for stem cells in the skin6. Interestingly, in human HSCs HR, not NHEJ, seems to be the repair mechanism of choice for double strand breaks3. Whether this functional difference between the two species is real or merely represents a technological or experimental difference remains to be seen.
A stem cell&#39;s repertoire to repair damaged DNA is likely to include other DNA repair mechanisms, such as base excision repair (BER), nucleotide excision repair (NER) and mismatch repair (MMR). BER and NER are responsible for repairing single or multiple base pair lesions in single stranded DNA, while MMR fixes base-base mismatches and insertion/deletion loops; these types of DNA damage cannot be repaired by NHEJ or HR. Supporting this notion are several studies from the hematopoietic system demonstrating a link between alterations in one of these pathways and abnormalities in the HSC compartment7-9, as well as an increased risk of developing myelodysplastic syndrome10-16, a disease that originates in the HSC and that is associated with increasing genomic instability as the disease progresses17. As of yet, measurements of BER, NER, and MMR directly in HSCs have not been reported.
In addition to elucidating the various processes that control tissue integrity at a mechanistic level, it is imperative to be able to measure the extent of mutated DNA, so that the consequences of aberrations in one of these processes can be tested, e.g. in normal versus genetically engineered stem cells or in old versus young. However, the development of a relevant assay is difficult because of the paucity of tissue-specific stem cells and the lack of culture conditions that preserves &#34;stemness&#34;. Moreover, such an assay should be amendable to environmental and genetic manipulations. A possible solution to these limitations and requirements is the use of mouse models that are specifically engineered to detect DNA mutations.
Multiple transgenic mouse models for mutation detection have been developed. For example, LacI transgenic mice18 carry a recoverable &#955; phage vector encoding the LacI reporter system, in which the LacI gene encodes a suppressor of the Lac operator and serves as the mutation reporter. Upon mutation of the LacI gene, the Lac operator is activated and &#946;-galactosidase is produced. &#946;-galactosidase cleaves the chromogenic substrate X-gal (5-bromo-4-chloro-e-indolyl-&#946;-D-galactopyranoside), which turns it blue. The cos sites flanking the LacI vector allows easy recovery by lambda phage proteins and subsequent infection of E. coli. After incubating infected E. coli on agarose that contains the X-gal substrate, plaques can be scored. Blue plaques contain a putative mutant Lac-I carrying phage, while clear plaques harbor non-mutants. The frequency of blue plaques (among the clear ones) indicates the mutant frequency in the original cell population the DNA was extracted from. Moreover, the &#955; phage hosting the LacI target can be readily sequenced using PCR techniques for relatively high throughput analysis. Sequencing multiple mutant LacI genes will reveal important information about the mutation spectrum, which in turn may point to possible deficiencies in specific DNA repair pathways or to specific genotoxic events. The LacI transgenic system has been standardized across multiple laboratories19 and the reagents are available commercially. One major disadvantage of the LacI system is the limited ability to detect large deletions or rearrangements; therefore, other methods, e.g. multi-color FISH on metaphase spreads need to be used to compliment this deficiency.
Within the &#955; phage vector of the LacI mouse model, there is a much smaller gene, CII, available for mutation analysis. Its size and the fact that mutants can be selected makes this a less labor-intensive and cheaper assay20 than the LacI gene analysis. However, the LacI gene is more extensively studied for mutagenesis21 and the sensitivity of the gene to mutations has been well characterized so that there is a clear understanding of the amino acid residues that generate a phenotypic response on a chromogenic substrate22-25.
Other mouse models for mutation detection include the use of the &#934;X174 or the LacZ transgenes. The &#934;X174 transgenic mouse model, with the original A:T&#8594;G:C reversion mutation assay26 or the forward mutation assay27 that allows detection of a spectrum of base pair substitutions, represents a less costly system than the LacI model. However, the mutational screen in the forward assay is not trivial and the mutation spectrum of the &#934;X174 transgene is not as well-characterized as that of the LacI. In mouse models carrying LacZ transgenes, the LacZ mutational reporter is recovered utilizing E. coli host cells that are sensitive to galactose and medium containing galactose28. A drawback of this system is that recovery of the LacZ target also involves restriction endonuclease digestion followed by ligation and electroporation of E. coli hosts, thereby making it difficult to adapt the system for small numbers of cells. Although it is not an absolute requirement for working with stem/progenitor cell populations (one can always start with more mice), if large numbers of cells are required (e.g. millions or more) it will quickly become impractical and cost-prohibitive. Also, the relatively large size of LacZ, while providing a sensitive mutational reporter, is cumbersome and more costly for DNA sequence analysis and determination of mutation spectra. A major advantage of this model however, is its ability to detect large deletions and insertions, as well as chromosomal rearrangements.
Since all cells in the LacI, &#934;X174 and LacZ transgenic mouse models carry the reporter system, any of these mouse models can be used to measure mutagenesis in any cell type of interest, including stem and progenitor cells, as long as they can be reliably harvested and in sufficient numbers. Because we had extensive experience with the LacI mouse model and the LacI mutation assay, we decided to pursue this system further for mutagenesis analysis in hematopoietic stem and progenitor populations.
The hematopoietic tissue is well-characterized in terms of cell surface phenotype of its individual components, including long-term repopulating stem cells, which are identifiable as the extremely rare population of Lin-IL7R-Sca-1+cKit++(LSK)/ Flk2-CD150+CD48- cells29. Mohrin et al.4 demonstrated that the slightly larger population of LSK/Flk2- cells are still good representatives for HSCs and significantly different from the most primitive committed myeloid progenitor (CMP) population when it comes to studying DNA repair. Moreover, when the HSC-enriched LSK (Flk-2+ and Flk-2-) cells were compared to the Lin-IL7R-Sca-1-cKit++ (LS-K progenitor cells), there was still a significant difference in NHEJ ability5 between the less pure, stem cell-enriched LSK population and the progenitor cells. In our study we use HSC-enriched LSK (Flk-2+ and Flk-2-) cells because we found that at least 2 x 105 cells are required for consistent, reliable results in this mutagenesis assay; this cell number is extremely difficult to obtain when one sorts the LSK/Flk2-CD150+CD48 population or even the LSK/Flk2- population (in terms of mice, costs and practicality). This protocol, based on the one originally developed by Kohler et al.18 describes in detail how the spontaneous DNA mutant frequency can be determined in LSK cells and defined populations of differentiated myeloid cells as well as unseparated bone marrow and spleen cells.

<table><tbody><tr><td>&lt;em&gt;LacI &lt;/em&gt;transgenic mice&amp;nbsp;</td><td>BioReliance Corporation</td><td></td><td></td></tr><tr><td>RecoverEase DNA Isolation Kit, including the RNace-It ribonuclease cocktail</td><td>Agilent Technologies (Stratagene)</td><td>720202</td><td></td></tr><tr><td>Transpack Packaging Extract, including the orange and blue transpack tubes and the SCS-8 &lt;em&gt;E. coli&lt;/em&gt;</td><td>Agilent Technologies (Stratagene)</td><td>200221</td><td></td></tr><tr><td>DNA size standards &amp;ndash; lambda ladder</td><td>Bio Rad</td><td>170-3635</td><td></td></tr><tr><td>0.025 mM Pore size membranes&amp;nbsp;</td><td>Fisher (Millipore)</td><td>VSWP 025 00</td><td></td></tr><tr><td>245 mm&lt;sup&gt;2&lt;/sup&gt; Bioassay dishes (trays)&amp;nbsp;</td><td>Fisher (Corning)</td><td>07-200-600</td><td></td></tr><tr><td>NZY broth (powder)</td><td>Fisher (Teknova)</td><td>N1144</td><td></td></tr><tr><td>Agar</td><td>Fisher</td><td>BP1423-500</td><td></td></tr><tr><td>Agarose</td><td>Fisher</td><td>E-3120-500</td><td></td></tr><tr><td>N,N-Dimethylformamide</td><td>Fisher</td><td>AC34843-5000</td><td></td></tr><tr><td>X-gal</td><td>Research Products Intl Corp (RPI)</td><td>B71800-10.0</td><td></td></tr><tr><td>Proteinase K</td><td>Roche</td><td>3-115-852</td><td></td></tr><tr><td>PCR Extender Taq Polimerase kit</td><td>5 PRIME</td><td>2200500</td><td></td></tr><tr><td>Agencourt AMPure XP cleanup kit</td><td>Beckman/Coulter</td><td>A63880</td><td></td></tr></tbody></table>

identifying dna mutations in purified hematopoietic stem/progenitor cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

The in vivo mutagenesis assay measures a rare event (mutant PFUs) among many events (all PFUs). By performing the assay with small number of cells, it is possible that the outcome is considerably influenced by false-positive and false-negative results. To address this issue we performed a serial dilution experiment with unfractionated bone marrow cells, harvested from three different animals. We measured the mutant frequency in the bone marrow of these animals using 1.4 x 106, 7.0 x 105, 3....

Watch this Scientific Journal Video about Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells at JoVE.com

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells | Immunology and Infection | Video

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

identifying-dna-mutations-purified-hematopoietic-stemprogenitor

Research

Education

JoVE Journal

JoVE Core

Biology

Immunology and Infection

Evolution

Population Genetics

SCIENTISTS IN ACTION

13.0K Views.  UT Health Science Center at San Antonio.  Hier beschrijven we een in vivo mutagenese test voor kleine aantallen hematopoietische cellen gezuiverd met de Lacl transgene muismodel. De Lacl gen kan worden geïsoleerd om de frequentie, locatie en soort DNA mutanten spontaan ontstaan ​​of na blootstelling aan genotoxinen bepalen. 

Video: Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and leukemogenesis. They have the capacity to differentiate into lymphoid and myeloid lineages. The colony forming cell (CFC) assay is used to study the proliferation and differentiation pattern of hematopoietic progenitors by their ability to form colonies in a semisolid medium. The number and the morphology of the colonies formed by a fixed number of input cells provide preliminary information about the ability of progenitors to differentiate and proliferate. Cells can be harvested from individual colonies or from the whole plate to further assess their numbers and differentiation states using flow cytometry and morphologic evaluation of Giemsa-stained slides. This assay is useful for assessing myeloid but not lymphoid differentiation. The term myeloid in this context is used in its wider sense to encompass granulocytic, monocytic, erythroid, and megakaryocytic lineages.

We have used this assay to assess the effects of oncogenes on the differentiation of primary human CD34+ cells derived from peripheral blood. For this purpose cells are transduced with either control retroviral construct or a construct expressing the oncogene of interest, in this case NUP98-HOXA9. We employ a commonly used retroviral vector, MSCV-IRES-GFP, that expresses a bicistronic mRNA that produces the gene of interest and a GFP marker. Cells are pre-activated by growing in the presence of cytokines for two days prior to retroviral transduction. After another two days, GFP+ cells are isolated by fluorescence-activated cell sorting (FACS) and mixed with a methylcellulose-containing semisolid medium supplemented with cytokines and incubated till colonies appear on the surface, typically 14 days. The number and morphology of the colonies are documented. Cells are then removed from the plates, washed, counted, and subjected to flow cytometry and morphologic examination. Flow cytometry with antibodies specific to the cell surface markers expressed during hematopoiesis provides information about lineage and maturation stage. Morphological studies of individual cells under a microscope after Wright- Giemsa staining provide further information with regard to lineage and maturation. Comparison of cells transduced with control empty vector to those transduced with an oncogene reveals the effects of the oncogene on hematopoietic differentiation.

Colony Forming Cell CFC Assay for Human Hematopoietic Cells

The colony forming cell (CFC) assay is an in vitro assay in which hematopoietic progenitors form colonies in a semi-solid medium. A combination of colony morphology, cell morphology, and flow cytometry are used to assess the ability of the progenitors to proliferate and differentiate along the different hematopoietic lineages.

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and ...

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is inherently a single-cell assay, however prior to molecular analysis, the target cells are often prospectively isolated in bulk, thereby losing single-cell resolution. Single-cell gene expression analysis provides a means to understand molecular differences between individual cells in heterogeneous cell populations. In bulk cell analysis an overrepresentation of a distinct cell type results in biases and occlusions of signals from rare cells with biological importance. By utilizing FACS index sorting coupled to single-cell gene expression analysis, populations can be investigated without the loss of single-cell resolution while cells with intermediate cell surface marker expression are also captured, enabling evaluation of the relevance of continuous surface marker expression. Here, we describe an approach that combines single-cell reverse transcription quantitative PCR (RT-qPCR) and FACS index sorting to simultaneously characterize the molecular and immunophenotypic heterogeneity within cell populations.
In contrast to single-cell RNA sequencing methods, the use of qPCR with specific target amplification allows for robust measurements of low-abundance transcripts with fewer dropouts, while it is not confounded by issues related to cell-to-cell variations in read depth. Moreover, by directly index-sorting single-cells into lysis buffer this method, allows for cDNA synthesis and specific target pre-amplification to be performed in one step as well as for correlation of subsequently derived molecular signatures with cell surface marker expression. The described approach has been developed to investigate hematopoietic single-cells, but have also been used successfully on other cell types.
In conclusion, the approach described herein allows for sensitive measurement of mRNA expression for a panel of pre-selected genes with the possibility to develop protocols for subsequent prospective isolation of molecularly distinct subpopulations.

Bulk gene expression measurements cloud individual cell differences in heterogeneous cell populations. Here, we describe a protocol for how single-cell gene expression analysis and index sorting by Florescence Activated Cell Sorting (FACS) can be combined to delineate heterogeneity and immunophenotypically characterize molecularly distinct cell populations.

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is ...

Genetics

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.

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 ...

Developmental Biology

Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data in all cells, including stem cells, and its integrity is fundamental to its regenerative ability. Stem cells undergo rapid propagation in labs to achieve the necessary numbers for transplantation. Accelerated cell growth leads to the loss of DNA integrity by accumulated metabolites, such as reactive oxygen, carbonyl, and alkylating agents. Transplanting these cells would result in poor engraftment and regeneration of the deteriorating organ. Moreover, transplanting DNA-damaged cells leads to mutations, DNA instability, cellular senescence, and possibly, life-threatening diseases such as cancer. Therefore, there is an immediate need for a quality control method to evaluate the cell's suitability for transplantation. Here, we provide step-by-step protocols for the assessment of the DNA integrity of stem cells prior to cell transplantation.

Here, we provide a detailed description of an experimental setup for an analysis of the assessment of DNA integrity in stem cells prior to cell transplantation.

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data ...

Characterizing Mutational Load and Clonal Composition of Human Blood

Hematopoietic stem and progenitor cells (HSPCs) gradually accumulate DNA mutations during a lifespan, which can contribute to age-associated diseases such as leukemia. Characterizing mutation accumulation can improve understanding of the etiology of age-associated diseases. Presented here is a method to catalogue somatic mutations in individual HSPCs, which is based on whole-genome sequencing (WGS) of clonal primary cell cultures. Mutations that are present in the original cell are shared by all cells in the clonal culture, whereas mutations acquired in vitro after cell sorting are present in a subset of cells. Therefore, this method allows for accurate detection of somatic mutations present in the genomes of individual HSPCs, which accumulate during life. These catalogues of somatic mutations can provide valuable insights into mutational processes active in the hematopoietic tissue and how these processes contribute to leukemogenesis. In addition, by assessing somatic mutations that are shared between multiple HSPCs of the same individual, clonal lineage relationships and population dynamics of blood populations can be determined. As this approach relies on in vitro expansion of single cells, the method is limited to hematopoietic cells with sufficient replicative potential.

Somatic mutation patterns in cells reflect previous mutagenic exposure and can reveal developmental lineage relationships. Presented here is a methodology to catalogue and analyze somatic mutations in individual hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cells (HSPCs) gradually accumulate DNA mutations during a lifespan, which can contribute to age-associated diseases such ...

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 ...

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 ...

Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional properties such as proliferation and differentiation, thereby promoting the development of hematologic malignancies. Efficient and precise genetic manipulation of HSPCs is required to model, characterize, and better understand the functional consequences of recurrent somatic mutations. Mutations can have a deleterious effect on a gene and result in loss-of-function (LOF) or, in stark contrast, may enhance function or even lead to novel characteristics of a particular gene, termed gain-of-function (GOF). In contrast to LOF mutations, GOF mutations almost exclusively occur in a heterozygous fashion. Current genome-editing protocols do not allow for the selective targeting of individual alleles, hampering the ability to model heterozygous GOF mutations. Here, we provide a detailed protocol on how to engineer heterozygous GOF hotspot mutations in human HSPCs by combining CRISPR/Cas9-mediated homology-directed repair and recombinant AAV6 technology for efficient DNA donor template transfer. Importantly, this strategy makes use of a dual fluorescent reporter system to allow for the tracking and purification of successfully heterozygously edited HSPCs. This strategy can be employed to precisely investigate how GOF mutations affect HSPC function and their progression toward hematological malignancies.

Novel strategies to faithfully model somatic mutations in hematopoietic stem and progenitor cells (HSPCs) are necessary to better study hematopoietic stem cell biology and hematological malignancies. Here, a protocol to model heterozygous gain-of-function mutations in HSPCs by combining the use of CRISPR/Cas9 and dual rAAV donor transduction is described.

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional ...

Cancer Research

Evaluation of Abnormal Growth-related Genes of Hematopoietic Stem and Progenitor Cells by Combining CRISPR/Cas9 Technology with Cell Counting

Hematopoietic stem cells possess the ability for long-term self-renewal and the potential to differentiate into various types of mature blood cells. However, the accumulation of cancerous mutations in hematopoietic stem and progenitor cells (HSPCs) can block normal differentiation, induce aberrant proliferation, and ultimately lead to leukemogenesis. To identify and/or evaluate the cancerous mutations, we integrated CRISPR/Cas9 technology with the Cell Counting Kit-8 (CCK-8) assay to investigate a model gene Trp53, that is essential for abnormal proliferative ability of HSPCs. Specifically, bone marrow cells enriched for HSPCs from Cas9 mice were harvested and then subjected to viral transfection with single-guide RNAs (sgRNAs) targeting one or several candidate genes to introduce genetic alterations in HSPCs. Then, a CCK-8 assay was performed to investigate the proliferative capacity of transfected HSPCs. The sgRNA targeting efficiency was confirmed by a Tracking of Indels by Decomposition assay. These identified genes may play crucial roles in leukemogenesis and could serve as potential therapeutic targets.

Here, we present a protocol that integrates CRISPR/Cas9 technology with the Cell Counting Kit-8 (CCK-8) assay to identify candidate genes that are crucial for the abnormal proliferative capacity of hematopoietic stem and progenitor cells.

Hematopoietic stem cells possess the ability for long-term self-renewal and the potential to differentiate into various types of mature blood cells. ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

Samenvatting

Meer video's verkennen