Isolation Of Hematopoietic Stem Cells Research Articles

Optimization of pre-enrichment strategies for mouse hematopoietic stem cell isolation and metabolomic analysis

Blood cell production arises from the activity of hematopoietic stem cells (HSCs), defined by their self-renewal capacity and ability to give rise to all mature blood cell types. The mouse remains one of the most studied species in hematological research, and markers to define and isolate mouse HSCs are well-established. Given the very low frequency of HSCs in the bone marrow, stem cell pre-enrichment by red blood cell lysis and magnetic cell separation is often performed as part of the isolation process to reduce sorting times. Several pre-enrichment strategies are available, differing in their speed, degree of enrichment, final cell yield, and cost. In the current study, we performed a side-by-side comparison and provide a decision tree to help researchers select a pre-enrichment strategy for mouse HSC isolation depending on their downstream application. We then compared different pre-enrichment techniques in combination with metabolomics analysis of HSCs, where speed, yield and temperature during pre-enrichment are crucial factors, and found that the choice of pre-enrichment strategy significantly impacts the number of metabolites detected and levels of individual metabolites in HSCs.

Isolation of Human Hematopoietic Stem Cells from an Apheresis Sample.

The hematopoietic system constantly produces new blood cells through hematopoiesis, and maintaining this balance is vital for human health. This balance is maintained by self-renewing hematopoietic stem cells (HSCs) and various progenitor cells. Under typical circumstances, HSCs are not abundantly found in peripheral blood; hence, their mobilization from the bone marrow is vital. Hematopoietic growth factors achieve this effectively, enabling mobilization and thus allowing blood sample and thus HSC collection via apheresis. Securing a sufficient supply of HSCs is vital for successful hematopoietic reconstitution and the rapid integration of committed cells. Thus, isolation and expansion of HSCs are crucial for convenient extraction, production of transplantable quantities, genetic modifications for enhanced therapeutic efficacy, and as a source of increased/expanded/synthesized blood cells in vitro. In conclusion, the isolation and expansion of HSCs play pivotal roles in both regenerative medicine and hematology. This protocol describes the isolation of human HSCs by providing an overview of the primary method for isolating human hematopoietic stem cells from apheresis blood samples and sheds light on human HSC studies and developments in research and medicine.

Exploring hematopoietic stem cell population in human milk and its benefits for infants: A scoping review

Objective: To comprehensively explore hematopoietic stem cells (HSCs) in human milk, understanding their molecular markers, isolation methods, benefits for infants, and potential medical applications. Methods: We conducted a scoping literature review following the PRISMA-ScR guidelines. This review included studies investigating HSCs in human milk, utilizing molecular markers such as CD34+, CD113+, and CD117+ for characterization. Both in vitro and in vivo studies exploring the morphology, function, and clinical implications of these cells were considered. The diverse range of papers reviewed were indexed in PubMed, Science Direct, Scopus, Sage Journals, and Google Scholar, published between 2010 and 2023. Results: This scoping review explored 577 articles and selected 13 studies based on our inclusion criteria, focusing on HSCs in human milk. Most studies dilute samples prior to HSC isolation, followed by detection using markers such as CD34+, CD113+, and CD117+, with flow cytometry serving as the primary analysis tool, focusing on their isolation and detection methods. While no definitive benefits have been conclusively established, there is a strong belief in the potential of HSCs to positively impact infant immunity, growth, and tissue repair. Conclusions: This review presents significant evidence supporting the presence of HSCs in human milk, identified by markers such as CD34+, CD113+, and CD117+. These cells show considerable potential in enhancing infant health, including immunity, tissue repair, cognitive development, and gastrointestinal health. Despite methodological variations in isolation and detection techniques, the collective findings underscore the potential clinical relevance of HSCs in human milk. Moreover, this review highlights the non-invasive accessibility of human milk as a source of HSCs and emphasizes the need for further research to unlock their therapeutic potential.

Combination of CD49b and CD229 Reveals a Subset of Multipotent Progenitors With Short-Term Activity Within the Hematopoietic Stem Cell Compartment.

Hematopoiesis is maintained by hematopoietic stem cells (HSCs) that replenish all blood lineages throughout life. It is well-established that the HSC pool is functionally heterogeneous consisting of cells differing in longevity, self-renewal ability, cell proliferation, and lineage differentiation. Although HSCs can be identified through the Lineage-Sca-1+c-Kit+CD48-CD34-CD150+ immunophenotype, the cell surface marker combination does not permit absolute purification of functional HSCs with long-term reconstituting ability. Therefore, prospective isolation of long-term HSCs is crucial for mechanistic understanding of the biological functions of HSCs and for resolving functional heterogeneity within the HSC population. Here, we show that the combination of CD229 and CD49b cell surface markers within the phenotypic HSC compartment identifies a subset of multipotent progenitor (MPP) cells with high proliferative activity and short-term reconstituting ability. Thus, the addition of CD229 and CD49b to conventional HSC markers permits prospective isolation of functional HSCs by distinguishing MPPs in the HSC compartment.

Optimizing of the isolation of hematopoietic stem cell in bone marrow transplantation in children

Most of collected hematopoietic stem cell (HSCs) products need processing in order to isolate stem cells, squeeze out of plasma and erythrocytes. There are two main aims for bone marrow (BM) enrichment: reduction of immunogenicity of AB0 incompatible transplants and/or preventing toxicity of hemolysis during cryopreservation. In our center we have implemented two methods for BM enrichment: manual technic using 10 % HAES (hydroxyethyl starch) and automatic cell separator. In order to optimize the process, we examined retrospectively the parameters which could have a great impact on final efficiency of engraftment, such as reduction of hematocrit, CD34 + , WBC recovery and cell viability. This study was a retrospective analysis of 46 pediatric patients (pts) who underwent autologous or allogeneic HSCT. There were performed 27 procedures using cell separator and 19 with HAES technique. This study showed that cell separator processing is significantly less damaging for stem cells than widely longer, manual HAES technique. Comparing RBC depletion and WBC recovery both used techniques are same efficient and good enough but we found out a significant difference in the efficiency of CD34 + recovery which was much higher in a technique of cell separator. We examined also the effect of addition of packed red blood cells (PRBCs) to the BM on purifying and efficiency of HSCs isolation. Doing so it decreased only the WBC recovery during sell separator processing. To sum up after series of analyzes we found out that cell separator is more convenient than HAES technique in most of considered aspects. Furthermore, cell separator use is cheaper and needs less time for processing.

Isolation and Characterization of Fetal Liver Hematopoietic Stem Cells.

Hematopoietic stem cells (HSCs) are responsible for the generation and maintenance of pools of multipotent precursors that ultimately give rise to all fully differentiated blood and immune cells. Proper identification and isolation of HSCs for functional analysis has greatly facilitated our understanding of both normal and abnormal adult hematopoiesis. Whereas adult hematopoiesis inmice and humans is driven by quiescent HSCs that reside almost exclusively within the bone marrow (BM), developmental hematopoiesis is characterized by a series of transient progenitors driving waves of increasingly mature hematopoietic cell production that occur across multiple anatomical sites. These waves of hematopoietic cell production are also responsible for the generation of distinct immune cell populations during development that persist into adulthood and contribute uniquely to adult immunity. Therefore, methods to properly isolate and characterize fetal progenitors with high purity across development become increasingly important not only for defining developmental hematopoietic pathways, but also for understanding the contribution of developmental hematopoiesis to the immune system. Here, we describe and discuss methods and considerations for the isolation and characterization of HSCs from the fetal liver, the primary hematopoietic organ during fetal development.

Hematopoietic Stem Cells Characteristics: From Isolation to Transplantation.

Hematopoietic stem cells (HSCs) have self-renewal as well as pluripotency properties and are responsible for producing all types of blood cells. These cells are generated during embryonic development and transit through various anatomical niches (bone marrow microenvironment). Today, they are easily enriched from some sources, including peripheral blood, bone marrow, and umbilical cord blood (UCB). HSCs have been used for many years to treat a variety of cancers and blood disorders such as various types of leukemia, lymphoma, myelodysplastic, myeloproliferative syndromes, etc. Although almost 50 years have passed since the discovery of stem cells and numerous investigations on cell therapy and regenerative medicine have been made, further studies need to be conducted in this regard. This manuscript review the history, location, evolution, isolation, and therapeutic approaches of HSCs.

Three-Dimensional Avian Hematopoietic Stem Cell Cultures as a Model for Studying Disease Pathogenesis.

Three-dimensional (3D) cell culture is attracting increasing attention today because it can mimic tissue environments and provide more realistic results than do conventional cell cultures. On the other hand, very little attention has been given to using 3D cell cultures in the field of avian cell biology. Although mimicking the bone marrow niche is a classic challenge of mammalian stem cell research, experiments have never been conducted in poultry on preparing in vitro the bone marrow niche. It is well known, however, that all diseases cause immunosuppression and target immune cells and their development. Hematopoietic stem cells (HSC) reside in the bone marrow and constitute a source for immune cells of lymphoid and myeloid origins. Disease prevention and control in poultry are facing new challenges, such as greater use of alternative breeding systems and expanding production of eggs and chicken meat in developing countries. Moreover, the COVID-19 pandemic will draw greater attention to the importance of disease management in poultry because poultry constitutes a rich source of zoonotic diseases. For these reasons, and because they will lead to a better understanding of disease pathogenesis, in vivo HSC niches for studying disease pathogenesis can be valuable tools for developing more effective disease prevention, diagnosis, and control. The main goal of this review is to summarize knowledge about avian hematopoietic cells, HSC niches, avian immunosuppressive diseases, and isolation of HSC, and the main part of the review is dedicated to using 3D cell cultures and their possible use for studying disease pathogenesis with practical examples. Therefore, this review can serve as a practical guide to support further preparation of 3D avian HSC niches to study the pathogenesis of avian diseases.

An Improved Lentiviral Fluorescent Genetic Barcoding Approach Distinguishes Hematopoietic Stem Cell Properties in Multiplexed In Vivo Experiments.

Hematopoietic stem cells (HSCs) represent a rare cell population of particular interest for biomedical research and regenerative medicine. Various marker combinations enable the isolation of HSCs, but fail to reach purity in transplantation assays. To reduce animal consumption, we developed a multiplexing system based on lentiviral fluorescent genetic barcoding (FGB) to enable the parallel characterization of multiple HSC samples within single animals. While previous FGB-mediated HSC multiplexing experiments achieved high in vitro gene marking rates, in vivo persistence of transduced cells remained suboptimal. Thus, we aimed to optimize vector design and gene transfer protocols to demonstrate the applicability of FGB for functional characterization of two highly similar HSC populations in a reduced number of mice. We developed a set of six new lentiviral FGB vectors, utilizing individual and combinatorial expression of Azami Green, mCherry, and YFP derivatives. Gene transfer rates were optimized by overnight transduction of prestimulated HSCs with titrated vector doses. Populations for competitive transplantation experiments were identified by immunophenotyping murine HSCs. This identified an LSK-SLAM- (Lin-Sca-1+cKit+CD48-CD150+EPCR-) cell subpopulation that lacks EPCR expression and exhibits prospectively reduced self-renewal potential compared with prototypical ESLAM (CD45+EPCR+CD48-CD150+) HSCs. We monitored 30 data points per HSC-subpopulation in two independent experiments (each n = 5) after cotransplantation of three uniquely color-coded ESLAM and LSK-SLAM- samples per recipient. While the first experiment was hampered by data fluctuations, increasing cell numbers and exchange of the internal promoter in the second experiment led to 74.4% chimerism, with 87.1% of fluorescent cells derived from ESLAM HSCs. Furthermore, ESLAM-derived cells produced 88.1% of myeloid cells, which is indicative of their origin from long-term repopulating HSCs. This work verifies the importance of EPCR for long-term repopulating HSCs and demonstrates the applicability of our optimized FGB-driven multiplexing approach for the efficient characterization of blood cell populations in biomedical research.

Isolation and <i> Ex Vivo</i> Expansion of Human Hematopoietic Stem Cells Derived from Umbilical Cord Blood

Umbilical cord blood (UCB) is a current major source of hematopoietic stem cells (HSCs) for cell transplantation therapy. Cell transplantation with HSCs derived from UCB is advantageous over transplantation with HSCs from adult tissues. However, the low number of HSC derived from a single unit of UCB limits its application. Thus, ex vivo expansion is a good option to create more UCB HSCs for clinical application. The strategies for HSC expansion in vitro focus on mimicking the composition and structure of HSC natural niche by enhancing self-renewal and inhibiting lineage differentiation of HSCs. In the past decade, the mechanisms of the interaction between HSC and the natural niche have been deeply investigated. This great progress in basic research has led to advancements in UCB HSC ex vivo expansion. In addition, the biological characteristics of the originally isolated UCB HSCs correlate with outcome of subsequent ex vivo expansion. In this paper, we summarize the late progress achieved in isolation and ex vivo expansion of UCB HSCs. Importantly, we attempt to provide an impact and practicable procedure to expand UCB HSC in vitro from isolation of original HSCs to identification of expanded HSCs.

Neutrophils generated in vitro from hematopoietic stem cells isolated from apheresis samples and umbilical cord blood form neutrophil extracellular traps.

Neutrophils release neutrophil extracellular traps (NET) comprising of decondensed chromatin that immobilizes and kills pathogens. In vitro generation of neutrophils on a large scale from hematopoietic stem cells (HSCs) may be a useful strategy for treating neutropenic patients in future, though it is not in clinical practice yet. Microbial infections lead to major cause of morbidity and mortality in these patients. Despite the importance of NET in preventing infection, efficacy of in vitro-generated neutrophils from HSCs to form NET is not tested. We show that functional neutrophils could be generated in vitro from HSCs/MNCs isolated from umbilical cord blood (UCB) and apheresis-derived peripheral blood (APBL). Neutrophils generated from UCB showed properties comparable to those isolated from peripheral blood. We also show that isolation of HSCs is not absolutely essential for in vitro neutrophil generation. Further, we show that neutrophils generated from HSCs express PADI4 enzyme and their NET-forming ability is comparable to peripheral blood neutrophils. Taken together, our data show that fully functional neutrophils can be generated in vitro from HSCs. NET-forming ability of in vitro-generated neutrophils is an important parameter to determine their functionality and thus, should be studied along with other standard functional assays.

Hematopoietic Stem Cell Cultures and Assays.

The adult hematopoietic system is repopulated in its entirety from a rare cell type known as hematopoietic stem cells (HSCs) that reside in the marrow space throughout the skeletal system. Here we describe the isolation and identification of HSCs both phenotypically and functionally.

Analysis and Isolation of Mouse Leukemic Stem Cells.

Flow cytometry has been widely used in basic and clinical research for analysis of a variety of normal and malignant cells. Hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs) can be highly purified by flow cytometry. Isolated HSCs and LSCs can be functionally identified by transplantation assays and can also be studied at the molecular level. Here we describe the flow cytometry methods for analysis and isolation of mouse HSCs and LSCs.

Immunophenotypic- and Molecular Analysis of Human Hematopoietic Stem and Progenitor Heterogeneity

Hematopoietic stem cells (HSCs) have the capacity to differentiate into all hematopoietic lineages and at the same time self-renew to maintain the HSC pool. HSCs have been thoroughly investigated using immunophenotypic-, molecular- and functional-analysis resulting in the development of protocols for high-purity prospective isolation of human HSCs. However, within the current state-of-the-art HSC populations, 90% of the cells lack stem cell activity, confounding molecular analysis of HSC function. Thus, identification of novel immunophenotypic markers to delineate the HSC population would improve our understanding of HSC biology. To identify cell-surface markers with the potential to discriminate between functionally different cells within the HSC population, we performed antibody screens measuring the expression of 340 markers on human cord blood (CB) and bone marrow (BM). Candidate markers that divide the HSC population were included in single-cell CITE-seq experiments together with conventional HSC and progenitor markers for combined analysis of immunophenotype and RNA sequencing. This allowed us to correlate the molecular signature of each single-cell with the expression of 40 cell-surface proteins in CD34+ and CD34+CD38- populations of fetal liver (FL), CB, young- and old BM. Following sequencing, the cells were clustered based on molecular signature. Fourteen distinct groups with HSC-, multipotent progenitor-, and early committed progenitor profiles were identified. To investigate how the molecularly defined groups correlate to established populations within CD34+ HSPCs, the surface marker expression from the CITE-seq experiment was included in the analysis. The immunophenotypically defined GMP, MEP and CMP populations showed high molecular heterogeneity with cells at different stages of differentiation. The immunophenotypic HSCs (CD38-CD90+CD45RA-) correlated with the molecularly defined HSC population with a 75.6% overlap. To find novel surface markers for prospective isolation of HSCs pseudo-time analysis was used, allowing for correlation of surface marker expression with differentiation status. Interestingly, both CD35 and CD11a correlated with differentiation, with CD35 expression decreasing and CD11a expression increasing with pseudo-time. These two novel HSC marker-candidates are currently being functionally validated by transplantation analysis. To compare the progenitor composition of CD34+ HSPCs at different stages of life, young BM was used as a baseline control. Interestingly, compared to young BM CB CD34+ cells contained a higher frequency of multipotent progenitor cells and a decreased proportion of committed progenitors. In contrast, old CD34+ BM was reduced in multipotent progenitor frequencies with a corresponding relative increase of committed progenitors. However, both CB and old BM showed similar proportions of molecularly defined HSCs as compared to young BM. These results indicate that ageing causes a depletion of the earliest hematopoietic progenitor populations while the HSC pool remains intact. Together, using single cell CITE-seq we can describe the immunophenotypic- and molecular-heterogeneity of the HSC and progenitor populations and identify two novel cell-surface marker candidates for prospective isolation of HSCs. Disclosures No relevant conflicts of interest to declare.

Safe and Efficient Peripheral Blood Stem Cell Collection in Patients with Sickle Cell Disease Using Plerixafor

Background: Hematopoietic stem cell (HSC) gene therapy is potentially curative for sickle cell disease (SCD) provided that a sufficient quantity of long-term engrafting CD34+ HSCs can be safely collected and infused. In patients with SCD, peripheral blood (PB) mobilization with granulocyte colony stimulating factor is contraindicated, and steady-state bone marrow (BM) harvesting is associated with suboptimal HSC quality and yield. Hence, agents that promote safe and effective peripheral HSC mobilization in SCD are required. Methods: This phase I multicenter study investigated the safety and efficacy of plerixafor mobilization (240 µg/kg) followed by apheresis, processing, and HSC enrichment in 15 adult subjects with SCD (NCT03226691). Hydroxyurea treatment was stopped at least two weeks prior to mobilization and all participants who were not receiving long-term transfusion therapy received prophylactic red blood cell exchange prior to plerixafor administration to achieve a target sickle Hb (HbS) <30%. The primary endpoint was efficient collection of PB HSCs (target 2.0x106 CD34+ cells/kg, minimum 1.5x106) after plerixafor mobilization without serious adverse events (SAEs). Collection products were assessed for total WBC, CD34+, CD3+, and CD19+ composition by flow cytometry. Spearman's correlation test was used to assess the relationship between baseline CD34/µL, total CD34+ cells/kg collected, and total blood volume processed. Findings: Fifteen participants with SCD (HbSS n=13, HbSC n=1, HbSβ+ n=1) who met inclusion criteria were enrolled at St. Jude Research Children's Hospital (n=3) or NIH (n=12) between July 2017 and February 2019. All participants except one successfully met the minimum target CD34+ cells/kg yield with 2 or fewer mobilization and apheresis procedures, with almost half the participants (n=7) achieving ≥5.0x106 CD34+ cells/kg. The total WBC count increased by an average of 3.2-fold over baseline (range 1.7-5.0) to an average peak WBC count of 26.5x103/µL (range 14.1-47.4), with counts returning to baseline within 1-2 days. Median total WBC, CD34+, CD19+, and CD3+ cells/kg in the final apheresis product after one (n=12) or two (n=3) collection procedures were 0.9 x109 WBC/kg (range 0.4-1.7), 4.5x106 CD34+ cells/kg (range 0.9-12.0), 3.4x108 CD19+ cells/kg (range 0.5-4.9), and 3.6x108 CD3+ cells/kg (range 1.7-7.3), respectively. There was a strong positive correlation between baseline CD34/µL and total CD34+ cells/kg (rs = 0.7776, 95% CI 0.446-0.9215, p = 0.001). Participants with the lowest pre-apheresis CD34 cell count generally underwent higher blood volume processing (rs = - 0.1443, 95% CI -0.6075 to 0.3921, p = 0.59) in an effort to achieve target yields. However, higher blood volume processing did not translate to higher total CD34+ cells/kg collection yields (rs = - 0.2104, 95% CI -0.6488 to 0.3328, p = 0.43). A median of 97% of positively selected CD34+ cells were CD34high (range 73.6-99.4%) compared to 1.3% CD34low (range 0.09-24.4%) phenotype suggesting enrichment for long-term engrafting HSCs. Seven grade III AEs (two non-pain and five pain related) and one grade IV AE (non-pain - hemolysis) occurred and each resolved with symptomatic treatment. Plerixafor mobilization was overall safe and in most cases generated CD34+ cell yields that were sufficient for both genetic modification and back-up allogeneic transplantation (median cell yield 4.2x106 CD34+ cells/kg). Interpretation: This first multi-institutional phase I study suggests the efficiency and safety of plerixafor mobilization and apheresis collection in 15 adult subjects with SCD. The primary endpoint to obtain a target of 2.0x106 CD34+ cells/kg from the PB was exceeded without SAEs. Importantly, immunophenotyping data suggest that for individuals with SCD, the plerixafor mobilized cell product is more enriched for long-term engrafting HSCs compared to CD34+ cells isolated from BM. Given the risks of general anesthesia and the low quality and yield of CD34+ cells harvested from the BM of individuals with SCD, plerixafor mobilization represents a safe and potentially superior alternative for HSC isolation. Disclosures Hankins: Novartis: Research Funding; ASPHO: Honoraria; NHLBI: Research Funding; LYNKS Foundation: Research Funding; NHLBI: Honoraria; National Committee for Quality Assurance: Consultancy; Global Blood Therapeutics: Research Funding; Bluebird Bio: Consultancy. Sharma:Vertex Pharmaceuticals: Other: Study PI; Doris Duke Foundation: Research Funding. Weiss:GlaxoSmithKline: Consultancy; Rubius INC: Consultancy; Esperian: Consultancy; Beam Therapeutics: Consultancy; Cellarity INC: Consultancy.

IMMUNOPHENOTYPIC AND MOLECULAR HETEROGENEITY ANALYSIS OF HUMAN HEMATOPOIETIC STEM AND PROGENITORS

Hematopoietic stem cells (HSCs) have the capacity to differentiate into all hematopoietic lineages, and at the same time self-renew to maintain the HSC pool. HSCs have been thoroughly investigated using immunophenotypic-, molecular- and functional-analysis resulting in the development of protocols for high-purity prospective isolation of human HSCs. However, within the current state-of-the-art HSC populations, 90% of the cells are not bona fide HSCs, confounding molecular analysis of HSCs. Thus, identification of novel immunophenotypic markers to delineate the HSC population would improve our understanding of HSC biology. To identify cell-surface markers with the potential to discriminate between functionally different cells within the HSC population, we performed antibody screens measuring the expression of 340 markers on human cord blood (CB) and bone marrow (BM). Candidate markers that divide the HSC population were included in CITE-seq experiments together with conventional HSC and progenitor markers for combined analysis of single-cell immunophenotype-/RNA sequencing analysis. This allowed us to correlate the molecular signature of each single-cell with expression of 40 cell-surface proteins, in CD34+ and CD34+CD38- populations of CB and BM. Following sequencing the cells were clustered based on molecular signature. Distinct groups with HSC, progenitor and early committed progenitor profiles were identified. Interestingly, comparison between CB and BM showed differences in progenitor proportions while the fraction of cells with HSC signature remained intact. Additionally, three novel HSC marker-candidates were discovered which are being validated. Together, using CITE-seq we can describe the immunophenotypic- and molecular-heterogeneity of the HSC and progenitor populations and identify three novel cell-surface marker candidates for prospective isolation of HSCs.

Differences in Cell Cycle Status Underlie Transcriptional Heterogeneity in the HSC Compartment

Hematopoietic stem cells (HSCs) are considered a heterogeneous cell population. To further resolve the HSC compartment, we characterized a retinoic acid (RA) reporter mouse line. Sub-fractionation of the HSC compartment in RA-CFP reporter mice demonstrated that RA-CFP-dim HSCs were largely non-proliferative and displayed superior engraftmentpotential in comparison with RA-CFP-bright HSCs.Gene expression analysis demonstrated higher expression of RA-target genes in RA-CFP-dim HSCs, in contrast to the RA-CFP reporter expression, but both RA-CFP-dim and RA-CFP-bright HSCs responded efficiently to RA invitro. Single-cell RNA sequencing (RNA-seq) of >1,200 HSCs showed that differences in cell cycle activity constituted the main driver of transcriptional heterogeneity in HSCs. Moreover, further analysis of the single-cell RNA-seq data revealed that stochastic low-level expression of distinct lineage-affiliated transcriptional programs is a common feature of HSCs. Collectively, this work demonstrates the utility of the RA-CFP reporter line as a tool for the isolation of superior HSCs.

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice.

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

Cobblestone Area-forming Cell Assay of Mouse Bone Marrow Hematopoietic Stem Cells.

Bone Marrow Hematopoietic Stem Cells (HSCs) require bone marrow microenvironment for their maintenance and proliferation. Culture of Bone Marrow Mesenchymal Stromal Cells (MSCs) provides appropriate environmental signals for HSCs survival in vitro. Here, we provide a detailed protocol that describes culture conditions for MSCs, flow cytometric isolation of HSCs from mouse bone marrow, and perform co-culture of MSCs and HSCs known as Cobblestone area-forming cell (CAFC) assay. Altogether, CAFC assays can be used as a high-throughput in vitro screening model where efforts are made to understand and develop therapies for complex bone marrow diseases. This protocol needs 3 to 4 weeks starting from culturing MSCs, isolating LSK cells (HSCs), and to performing limited dilution CAFC assay.

ALDHs in normal and malignant hematopoietic cells: Potential new avenues for treatment of AML and other blood cancers

Multiple studies have demonstrated that ALDH1A1 is elevated in hematopoietic stem cells (HSCs). As a means to better characterize such cells, we previously developed the fluorescent ALDH1A1 substrate Aldefluor to facilitate HSC identification and isolation. This has proven useful for counting and isolating HSCs from human bone marrow, peripheral blood and cord blood as well as stem cells in other tissues and organisms. Given the high level expression of ALDH1A1, we explored its biology and that of other ALDHs in HSCs and found that ALDH1A1 and ALDH3A1 were important in metabolizing reactive aldehydes (RAlds) and reactive oxygen species (ROS). In murine models, loss of these two isoforms resulted in a variety of effects on HSC biology, increased DNA damage and predisposition to leukemia formation when combined with a genetic driver of HSC proliferation and self-renewal. Loss of ALDH activity may also predispose to marrow failure and AML in Fanconi's anemia (FA). ALDHs also have importance in mediating drug resistance in AML, may be useful in the identification of leukemia stem cells (LSCs) and ALDH activity levels may have prognostic significance. Together these findings suggest that further studying ALDH biology in AML and other blood cancers may provide important insights into malignant transformation and may point the way to the development of novel diagnostics and therapies.