Human hematopoietic microenvironments

Dormancy of hematopoietic stem cells and formation of progenitors are directed by signals that come from the bone marrow microenvironment. Considerable knowledge has been gained on the murine hematopoietic stem cell microenvironment, while less so on the murine progenitor microenvironment and even less so on these microenvironments in humans. Characterization of these microenvironments is decisive for understanding hematopoiesis and finding new treatment modalities against bone marrow malignancies in the clinic. However, it is equally challenging, because hematopoietic stem cells are difficult to detect in the complex bone marrow landscape. In the present study we are characterizing the human hematopoietic stem cell and progenitor microenvironment. We obtained three adjacent bone marrow sections from ten healthy volunteers. One was used to identify a population of CD34+/CD38- “hematopoietic stem cells and multipotent progenitors” and a population of CD34+/CD38+ “progenitors” based on immunofluorescence pattern/intensity and cellular morphology. The other two were immunostained respectively for CD34/CD56 and for CD34/SMA. Using the combined information we performed a non-computer-assisted quantification of nine bone marrow components (adipocytes, megakaryocytes, bone surfaces, four different vessel types (arteries, capillaries, sinusoids and collecting sinuses), other “hematopoietic stem cells and multipotent progenitors” and other “progenitors”) within 30 μm of “hematopoietic stem cells and multipotent progenitors”, “progenitors”, and “random cell profiles”. We show that the microenvironment of the “hematopoietic stem cells and multipotent progenitors” is significantly enriched in sinusoids and megakaryocytes, while the microenvironment of the “progenitors” is significantly enriched in capillaries, other “progenitors”, bone surfaces and arteries.

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Osteoblast ablation reduces normal long-term hematopoietic stem cell self-renewal but accelerates leukemia development

Blood ◽

10.1182/blood-2014-06-582924 ◽

2015 ◽

Vol 125 (17) ◽

pp. 2678-2688 ◽

Cited By ~ 64

Author(s):

Marisa Bowers ◽

Bin Zhang ◽

Yinwei Ho ◽

Puneet Agarwal ◽

Ching-Cheng Chen ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem Cell ◽

Self Renewal ◽

Hematopoietic Stem ◽

Key Points ◽

Leukemia Development

Key Points Bone marrow OB ablation leads to reduced quiescence, long-term engraftment, and self-renewal capacity of hematopoietic stem cells. Significantly accelerated leukemia development and reduced survival are seen in transgenic BCR-ABL mice following OB ablation.

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Phenotypic analysis of mouse hematopoietic stem cells shows a Thy-1- negative subset

Blood ◽

10.1182/blood.v80.8.1957.bloodjournal8081957 ◽

1992 ◽

Vol 80 (8) ◽

pp. 1957-1964 ◽

Cited By ~ 2

Author(s):

GJ Spangrude ◽

DM Brooks

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem Cell ◽

Bone Marrow Cells ◽

Cell Activity ◽

Mouse Strains ◽

Hematopoietic Stem ◽

Stem Cell Activity

Mouse hematopoietic stem cells can be identified and enriched from populations of normal bone marrow cells by immunofluorescent labeling of cell surface molecules followed by flow cytometric separation. We show here that the majority of hematopoietic stem cell activity, as defined by long-term competitive repopulation of irradiated animals and by a secondary transplant assay for spleen colony-forming units (CFU- S), could be localized in Ly-6b haplotype mice to a fraction of bone marrow cells that expresses the Ly-6A/E (Sca-1) molecule. Further, an analysis of hematopoietic stem cell activity in bone marrow of mouse strains expressing the Thy-1.1 allele indicated that the vast majority of activity was included in the Thy-1low population. In contrast, hematopoietic stem cell activity found in the bone marrow of Thy-1.2 genotype mouse strains was recovered in both the Thy-1neg and the Thy- 1low populations. However, similar to Thy-1.1 strains, most activity was localized to the Ly-6A/E+ population of cells. The difference in Thy-1 phenotype of hematopoietic stem cell activity apparent between Thy-1.1- and Thy-1.2-expressing mouse strains was not caused by differences in the staining intensity of monoclonal antibodies (MoAbs) specific for the Thy-1 alleles. Furthermore, an antiframework MoAb that stains both alleles of Thy-1 separated hematopoietic stem cell activity from mice expressing the two alleles in the same manner as did allele- specific MoAb. The results of this study show that Thy-1 expression is not an invariant characteristic of mouse hematopoietic stem cells, and that mice expressing the Thy-1.1 allele are unique in that hematopoietic stem cell activity is found exclusively in the Thy-1low population.

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DNA Microarray Analysis of GPI-Negative Hematopoietic Stem Cells in Paroxysmal Nocturnal Hemoglobinuria Patients.

Blood ◽

10.1182/blood.v106.11.1048.1048 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1048-1048

Author(s):

Kazuhiko Ikeda ◽

Tsutomu Shichishima ◽

Yoshihiro Yamashita ◽

Yukio Maruyama ◽

Hiroyuki Mano

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem Cell ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Bone Marrow Failure ◽

Data Set ◽

Hematopoietic Stem ◽

Pnh Clone

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematological disorder which is manifested by complement-mediated hemolysis, venous thrombosis, and bone marrow failure. Deficiencies of glycosylphosphatidylinositol (GPI)-anchored proteins, due to mutations in the phosphatidylinositol glycan-class A (PIG-A) gene, contribute to complement-mediated hemolysis and affect all hematopoietic lineages in PNH. However, it is unclear how a PNH clone with a PIG-A gene mutation expands in bone marrow. Although some genes, including the Wilms’ tumor gene (Shichishima et al, Blood, 2002), the early growth response gene, anti-apoptosis genes, and the gene localized at breakpoints of chromosome 12, have been reported as candidate genes that may associate with proliferations of a GPI-negative PNH clone, previous studies were not intended for hematopoietic stem cell, indicating that the differences in gene expressions between GPI-negative PNH clones and GPI-positive cells from PNH patients remain unclear at the level of hematopoietic stem cell. To identify genes contributing to the expansion of a PNH clone, here we compared the gene expression profiles between GPI-negative and GPI-positive fractions among AC133-positive hematopoietic stem cells (HSCs). By using the FACSVantage (Becton Dickinson, San Jose, CA) cell sorting system, both of CD59+AC133+ and CD59− AC133+ cells were purified from bone marrow mononuclear cells obtained from 11 individuals with PNH. Total RNA was isolated from each specimen with the use of RNeasy Mini column (Qiagen, Valencia, CA). The mRNA fractions were amplified, and were used to generate biotin-labeled cDNAs by the Ovation Biotin system (NuGEN Technologies, San Carlos, CA). The resultant cDNAs were hybridized with a high-density oligonucleotide microarray (HGU133A; Affymetrix, Santa Clara, CA). A total of >22,000 probe sets (corresponding to >14,000 human genes) were assayed in each experiment, and thier expression intensities were analyzed by GeneSpring 7.0 software (Silicon Genetics, Redwood, CA). Comparison between CD59-negative and CD59-positive HSCs has identified a number of genes, expression level of which was statistically different (t-test, P <0.001) between the two fractions. Interestingly, one of the CD59− -specific genes isolated in our data set turned out to encode a key component of the proteasome complex. On the other hand, a set of transcriptional factors were specifically silenced in the CD59− HSCs. These data indicate that affected CD59-negative stem cells have a specific molecular signature which is distinct from that for the differentiation level-matched normal HSCs. Our data should pave a way toward the molecular understanding of PNH.

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Murine hematopoietic stem cell characterization and its regulation in BM transplantation

Blood ◽

10.1182/blood.v96.9.3016.h8003016_3016_3022 ◽

2000 ◽

Vol 96 (9) ◽

pp. 3016-3022 ◽

Cited By ~ 3

Author(s):

Yi Zhao ◽

Yuanguang Lin ◽

Yuxia Zhan ◽

Gengjie Yang ◽

Jeffrey Louie ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Cell Sorting ◽

Cell Subset ◽

Transplant Recipients ◽

Hematopoietic Stem ◽

Murine Hematopoietic Stem Cell

Using 5-color fluorescence-activated cell sorting, we isolated a subset of murine pluripotent hematopoietic stem cells (PHSC) with the phenotype Lin− Sca+ kit+CD38+ CD34− that appears to fulfill the criteria for most primitive PHSC. In the presence of whole bone marrow (BM) competitor cells, these cells produced reconstitution in lethally irradiated primary, secondary, and tertiary murine transplant recipients over the long term. However, these cells alone could not produce reconstitution in lethally irradiated recipients. Rapid proliferation of these cells after BM transplantation required the assistance of another BM cell subset, which has the phenotype Lin− Sca+ kit+ CD38−CD34+.

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Hematopoietic Stem Cell Expansion by HOXB4 Is Greatly Enhanced in p21 Deficient Stem Cells.

Blood ◽

10.1182/blood.v104.11.1688.1688 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1688-1688 ◽

Cited By ~ 1

Author(s):

Noriko Miyake ◽

Ann C.M. Brun ◽

Mattias Magnusson ◽

David T. Scadden ◽

Stefan Karlsson

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem Cell ◽

Bone Marrow Cells ◽

Ex Vivo ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Hox transcription factors have emerged as important regulators of hematopoiesis. In particular, enforced expression of HOXB4 is a potent stimulus for murine hematopoietic stem cell (HSC) self-renewal. Murine HSCs engineered to overexpress HoxB4 expand significantly more than control cells in vivo and ex vivo while maintaining a normal differentiation program. HSCs are regulated by the cell proliferation machinery that is intrinsically controlled by cyclin-dependent kinase inhibitors such as p21Cip1/Waf1(p21) and p27Kip1 (p27). The p21 protein restricts cell cycling of the hematopoietic stem cell pool and maintains hematopoietic stem cell quiescence. In order to ask whether enhanced proliferation due to HOXB4 overexpression is mediated through suppression of p21 we overexpressed HOXB4 in hematopoietic cells from p21−/− mice. First, we investigated whether human HOXB4 enhances in vitro expansion of BM cells from p21−/− mice compared to p21+/+ mice. 5FU treated BM cells from p21−/− or p21+/+ mice were pre-stimulated with SCF, IL-6, IL-3 for 2 days followed by transduction using retroviral vector expressing HOXB4 together with GFP (MIGB4) or the control GFP vector (MIG). The cells were maintained in suspension cultures for 13 days and analyzed for GFP positive cells by flow-cytometry. Compared to MIG transduced BM cells from p21+/+ mice (MIG/p21+), the numbers of GFP positive cells were increased 1.1-fold in MIG/p21−, 3.2-fold in MIGB4/p21+ and 10.0-fold in MIGB4/p21− respectively (n=5). CFU assays were performed after 13 days of culture. The numbers of CFU were increased 4.8-fold in MIG/p21−, 19.5-fold in MIG/p21+ and 33.9 -fold in MIGB4/p21− respectively. Next, we evaluated level of HSCs expansion by bone marrow repopulation assays. After 12-days of culture, 1.5 x 105 MIGB4 or MIG-transduced cells (Ly5.2) were transplanted into lethally irradiated mice in combination with 8 x 105 fresh Ly5.1 bone marrow cells. Sixteen weeks after transplantation, no Ly5.2 cells could be detected in MIG/p21+ or MIG/p21− transplanted mice (n=6). In contrast, Ly5.2 positive cells were detected in both MIGB4/p21+/+ and MIGB4/p21−/− cells. The % of Ly5.2 positive cells in MIGB4/p21− transplanted mice was 9.9-fold higher than MIGB4/p21+ transplanted mice. (38.4 % vs 3.9 %, P<0.02, n=5). These Ly5.2 positive cells differentiated into all lineages, as determined by proportions of Mac-1, B-220, CD3 and Ter119 positive populations. Currently, we are enumerating the expansion of HOXB4 transduced HSCs in p21 deficient BM cells using the CRU assay. Our findings suggest that HOXB4 increases the self-renewal of hematopoietic stem cells by a mechanism that is independent of p21. In addition, the findings demonstrate that deficiency of p21 in combination with enforced expression of HOXB4 can be used to rapidly and effectively expand hematopoietic stem cells.

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Genetic Uncoupling of the Regulation of Hematopoietic Stem Cell Quiescence and Self-Renewal.

Blood ◽

10.1182/blood.v108.11.1347.1347 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1347-1347

Author(s):

Yan Liu ◽

Yasuhiko Miyata ◽

Goro Sashida ◽

Anthony Debalsio ◽

Yuhui Liu ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem Cell ◽

Quiescent State ◽

Double Knockout ◽

Ki 67 ◽

Self Renewal ◽

Hematopoietic Stem

Abstract It is usually stated that HSCs must choose to either self-renew or to differentiate and lose some of their multi potentiality. Recently, we demonstrated that MEF, an ETS family of transcription factor, played an important role in regulating HSC quiescence, illustrating a third choice for the HSC, namely to make an “active” choice and remain quiescent, without undergoing either self-renewal, or differentiation. MEF null HSCs are more quiescent than normal HSCs. In addition, MEF null mice exhibit greater numbers of hematopoietic stem cells and show resistance to chemotherapy and radiation. Little is known about the regulation of self-renewal vs. quiescence of HSCs, however the cdk inhibitor p21 has been implicated in regulating both HSC quiescence and proliferation. In the absence of p21, hematopoietic stem cell numbers are reported to be increased, but so is proliferation, leading to stem cell exhaustion. This implies that while p21 may maintain HSCs in their quiescent state, MEF functions to facilitate the entry of quiescent HSCs into the cycle, To investigate the potential opposing roles of MEF and p21 in HSC quiescence and self-renewal and to test whether the quiescent state of MEF null HSCs is dependent on the presence of p21, we have generated MEF / p21 double-knockout (DKO) mice. These mice are viable and born at normal mendelian frequency. MEF / p21 DKO mice have a higher than normal proportion of HSCs in the G0 phase, based on Pyronin Y/Hoechst staining and staining for the proliferation antigen Ki-67. Thus, the increased quiescence is not dependent on the presence of p21. However, by measuring LSK cells, we have observed a normal number of HSCs in the bone marrow of MEF / p21 DKO mice, in contrast to the increased number of HSCs in the bone marrow of MEF null mice. This suggests that the increased number of hematopoietic stem cells in MEF null mice is dependent on p21. Ongoing studies will further address the unique mechanisms that control HSC vs. stem cell expansion.

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Effect of Umbilical Cord Blood Hematopoietic Stem Cells on Formation of Angiogenic Structures.

Blood ◽

10.1182/blood.v110.11.4054.4054 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4054-4054

Author(s):

Aaron Victor ◽

Mary J. Laughlin ◽

Marcie R. Finney ◽

Nicholas J. Greco

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Coronary Artery ◽

Hematopoietic Stem Cells ◽

Cord Blood ◽

Hematopoietic Stem Cell ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Hematopoietic Stem

Abstract There is a significant unmet need for novel therapeutic treatments for patients presenting with chronic ischemic conditions such as coronary artery disease and diabetes. Revascularization measures, such as infusions with endothelial progenitor cells (EPC) characterized by the expression of early hematopoietic stem cell markers, hold significant potential in treating these patients. Pre-clinical and clinical studies using transplanted EPC to restore blood flow and improve cardiac function in animal models of ischemia have proven effective. Recent studies have used bone marrow mononuclear cells while some more recent studies have focused on enriched stem cell treatments, such as purified bone marrow hematopoietic stem cell (HSC) CD34+/133+ cell populations, in patients with coronary artery ischemia. In this study, the hypothesis to be tested was that umbilical cord blood-derived hematopoietic stem cells (CD34+/CD133+) cells may augment the formation and stability of angiogenic networks of cord-like structures derived from umbilical vein endothelial cells (HUVEC) cultured in growth factor-reduced Matrigel (GFR MG) assays. Umbilical cord blood MNC were isolated with ficoll and separated into HSC CD34+/133+ and CD34−/133− fractions. Positive fractions were flow cytometry, sorted for HSC, and stained with the lipophilic fluorescent red dye CM-DiI and the HUVEC were stained with the lipophilic fluorescent green dye Oregon Green. HUVEC alone or HSC and HUVEC were then co-cultured under hypoxic conditions (1% O2) on the GFR MG in 96 well plates. Cells were photographed with a fluorescent microscope at 16, 48, and 72 hours. Transwell experiments (0.4μm pores) were also performed with HSC CD34+/133+ and CD34−/133− fractions prepared and suspended in transwells above HUVEC plated on GFR MG on bottom wells. The presence of both HSC CD34+/133+ and CD34−/133− fractions increased the numbers of nodes (branch points of structures) and allowed the structures to persist when observed over three days (a representative experiment of N =3) (Table): Day 1 Day 1 Day 2 Day 2 Day 3 Day 3 Node # % Total Node # % Total Node # % Total HUVEC 11.6 ± 4.9 100 1.3 ± 1.2 9.2 0.33 ± 0.58 2.2 HUVEC + HSC CD34+/133+ 17.3 ± 9.2 100 6.3 ± 4.5 35.3 4.7 ± 5.5 21.4 HUVEC + HSC CD34−/133− 34 ± 13.2 100 19.7 ± 2.5 61.6 10 ± 3.6 29.8 The HSC CD34−/133− fraction resulted in a greater increase in node formation than the HSC CD34+/133+ and both fractions stimulated significant persistence in formed structures. In addition, CM-Dil labeled cells were localized at nodes points. Results with the transwell assay demonstrated that when either HSC CD34+/133+ or CD34−/133− fractions were suspended above HUVEC, augmentation of the formation of cord-like structures was not observed. In summary, both umbilical cord blood-derived HSC CD34+/133+ and CD34−/133− fractions possess properties that augment the formation of angiogenic structures. We observed that the number of nodes are greater in the presence of both HSC CD34+/133+ and CD34−/133− fractions than with HUVEC alone. The transwell experiment suggested that cell-to-cell interactions are necessary for augmentation of the cord structures. In future studies, we will address the mechanism of intercellular interactions that result in the augmentation of cord-like structures and which particular subpopulations within cord blood, both from HSC CD34+/133+ and CD34−/133− fractions are required for augmentation of structure formation.

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Critical Role Of Casein Kinase (Ck)1α Heterozygote Gene Inactivation In The Clonal Advantage Of Hematopoietic Stem Cells In Del(5q) MDS

Blood ◽

10.1182/blood.v122.21.98.98 ◽

2013 ◽

Vol 122 (21) ◽

pp. 98-98

Author(s):

Rebekka K. Schneider ◽

Dirk Heckl ◽

Marcus Järås ◽

Lisa Chu ◽

McConkey Marie ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

Hematopoietic Stem Cell ◽

Hematopoietic Stem ◽

Myeloid Progenitor Cells ◽

Stem And Progenitor Cells ◽

A Cell

Abstract Casein kinase 1α (Ck1α) is a serine/threonine kinase located in the common deleted region (5q32) in del(5q) myelodysplastic syndrome (MDS). Ck1α is a regulator of the canonical WNT signaling pathway and may play a role in the clonal advantage of del(5q) cells. In addition, we identified CK1α as a therapeutic target in myeloid malignancies in an in vivo RNA interference screen, and haploinsufficiency for CK1α could further sensitize del(5q) cells to CK1α inhibition. To explore the biology and therapeutic potential of CK1α in MDS, we generated a conditional Ck1α knockout mouse model. Conditional homozygous inactivation of Ck1α resulted in bone marrow failure, ablation of hematopoietic stem and progenitor cells, a severe anemia and rapid lethality within 7-12 days, confirming that Ck1α is essential for hematopoietic stem and progenitor cell survival. In contrast, mice with haploinsufficiency of Ck1α developed a hypercellular bone marrow, as is typical in MDS, a significantly elevated white blood cell count (p=0.002) and normal hemoglobin levels. The hematopoietic stem cells (LSK, LT-HSC, ST-HSC) as well as progenitor cells (LK, pre-GMP, GMP, pre-CFU-e, CFU-e, pre-megakaryocytes-erythrocytes) were not affected by Ck1α haploinsufficiency 14 days after induction. Only the megakaryocytic progenitor cells (p=0.04) were significantly reduced. This finding was in line with severe dysplasia and hypolobulated micromegakaryocytes observed in the bone marrow, another typical histomorphological feature of del(5q) MDS. In long-term experiments up to 8 months, the survival of mice with Ck1α haploinsufficiency was not impaired, although we observed an exhaustion of the stem cell pool with significant reduction of ST-HSC (p<0.001), LT-HSC (p=0.003), and MPP (p=0.007). We were able to demonstrate that this significant reduction is a cell-extrinsic effect. In transplantation and HSC repopulation assays, an intact HSC function and even a significant expansion of hematopoietic stem cells and progenitor cells with Ck1α haploinsufficiency was confirmed in comparison to MxCre controls (LSK p=0.019; LK p=0.035; CMP p=0.036; GMP p=0.027; MEP p=0.005), suggesting a repopulation advantage of HSC with Ck1α haploinsufficiency. In contrast, Ck1α homozygous deletion leads to a cell-autonomous, p53-mediated HSC failure in transplantation assays. To dissect the mechanism of hematopoietic stem cell expansion in Ck1α haploinsufficiency on the one hand and the hematopoietic stem cell ablation after Ck1α ablation on the other hand, we analyzed regulatory mechanisms including proliferation and apoptosis in LK cells (myeloid progenitor cells) and LSK cells (enriched for hematopoietic stem cells). Ablation of Ck1α led to a significant increase (p=0.001) in the number of LSK and LK in the S/M/G2 phase, accompanied by a significant reduction in the G0/G1 fraction, suggesting their exit from quiescence. Ck1α haploinsufficiency led to a significant increase in the fraction of cycling cells in myeloid progenitor cells (LK, p=0.052), the quiescent hematopoietic stem cells were not significantly affected. In Western Blots of ckit+ hematopoietic stem and progenitor cells, a significant increase of intracellular ß-catenin levels was detected in both Ck1α haploinsufficient and even stronger in Ck1α ablated cells, accompanied by an exit from stem cell quiescence shown by loss of p21-mediated growth arrest and up-regulation of phosphorylated retinoblastoma protein indicating cell cycle progression from G0 to G1 in comparison to the MxCre+ control cells. Ck1α ablation led to p53-mediated apoptosis in stem and progenitor cells (Annexin V/7-AAD). In Ck1α haploinsufficient cells, apoptosis was not significantly induced in neither LK cells or in LSK cells although p53 induction was observed in the bone marrow. Taken together, our results indicate that Ck1α is essential for hematopoietic stem and progenitor cell survival, but that Ck1α haploinsufficiency does not decrease, and may increase, hematopoietic stem cell function. This finding highlights the potential of preferential elimination of the del(5q) hematopoietic stem cells through Ck1α inhibtion and thus provides a potential therapeutic window. Consistent with this hypothesis, targeting the haploinsufficient kinase activity in vitro with the Ck1α small molecule inhibitor D4476, selectively targets CK1α haploinsufficient cells relative to wild-type cells. Disclosures: Järås: Cantargia: Equity Ownership.

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Phenotypic analysis of mouse hematopoietic stem cells shows a Thy-1- negative subset

Blood ◽

10.1182/blood.v80.8.1957.1957 ◽

1992 ◽

Vol 80 (8) ◽

pp. 1957-1964 ◽

Cited By ~ 37

Author(s):

GJ Spangrude ◽

DM Brooks

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem Cell ◽

Bone Marrow Cells ◽

Cell Activity ◽

Mouse Strains ◽

Hematopoietic Stem ◽

Stem Cell Activity

Abstract Mouse hematopoietic stem cells can be identified and enriched from populations of normal bone marrow cells by immunofluorescent labeling of cell surface molecules followed by flow cytometric separation. We show here that the majority of hematopoietic stem cell activity, as defined by long-term competitive repopulation of irradiated animals and by a secondary transplant assay for spleen colony-forming units (CFU- S), could be localized in Ly-6b haplotype mice to a fraction of bone marrow cells that expresses the Ly-6A/E (Sca-1) molecule. Further, an analysis of hematopoietic stem cell activity in bone marrow of mouse strains expressing the Thy-1.1 allele indicated that the vast majority of activity was included in the Thy-1low population. In contrast, hematopoietic stem cell activity found in the bone marrow of Thy-1.2 genotype mouse strains was recovered in both the Thy-1neg and the Thy- 1low populations. However, similar to Thy-1.1 strains, most activity was localized to the Ly-6A/E+ population of cells. The difference in Thy-1 phenotype of hematopoietic stem cell activity apparent between Thy-1.1- and Thy-1.2-expressing mouse strains was not caused by differences in the staining intensity of monoclonal antibodies (MoAbs) specific for the Thy-1 alleles. Furthermore, an antiframework MoAb that stains both alleles of Thy-1 separated hematopoietic stem cell activity from mice expressing the two alleles in the same manner as did allele- specific MoAb. The results of this study show that Thy-1 expression is not an invariant characteristic of mouse hematopoietic stem cells, and that mice expressing the Thy-1.1 allele are unique in that hematopoietic stem cell activity is found exclusively in the Thy-1low population.

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Connecting secretome to hematopoietic stem cell phenotype shifts in an engineered bone marrow niche

10.1101/2020.01.19.911800 ◽

2020 ◽

Cited By ~ 1

Author(s):

Aidan E. Gilchrist ◽

Brendan A.C. Harley

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

Hematopoietic Stem Cell ◽

Bone Marrow Niche ◽

Soluble Factors ◽

Hematopoietic Stem ◽

Secreted Factors

AbstractHematopoietic stem cells (HSCs) primarily reside in the bone marrow, where they receive external cues from their local microenvironment. The complex milieu of biophysical cues, cellular components, and cell-secreted factors regulates the process by which HSC produce the blood and immune system. We previously showed direct co-culture of primary murine hematopoietic stem and progenitor cells with a population of marrow-derived mesenchymal stromal and progenitor cells (MSPCs) in a methacrylamide-functionalized gelatin (GelMA) hydrogel improves hematopoietic progenitor maintenance. However, the mechanism by which MSPCs influenced HSC fate decisions remained unknown. Herein, we report the use of proteomic analysis to correlate HSC phenotype to a broad candidate pool of 200 soluble factors produced by combined mesenchymal and hematopoietic progeny. Partial Least Squares Regression (PLSR), along with an iterative filter method, identified TGFβ-1, MMP-3, c-RP, and TROY as positively correlated with HSC maintenance. Experimentally, we then observe exogenous stimulation of HSC monocultures in GelMA hydrogels with these combined cytokines increases the ratio of hematopoietic progenitors to committed progeny after a 7-day culture 7.52 ± 3.65 fold compared to non-stimulated monocultures. Findings suggest a cocktail of the downselected cytokines amplify hematopoietic maintenance potential of HSCs beyond that of MSPC-secreted factors alone. This work integrates empirical and computation methods to identify cytokine combinations to improve HSC maintenance within an engineered HSC niche, suggesting a route towards identifying feeder-free culture platforms for HSC expansion.InsightHematopoietic stem cells within an artificial niche receive maintenance cues in the form of soluble factors from hematopoietic and mesenchymal progeny. Applying a proteomic regression analysis, we identify a reduced set of soluble factors correlated to maintenance of a hematopoietic phenotype during culture in a biomaterial model of the bone marrow niche. We identify a minimum factor cocktail that promotes hematopoietic maintenance potential in a gelatin-based culture, regardless of the presence of mesenchymal feeder-cells. By combining empirical and computational methods, we report an experimentally feasible number of factors from a large dataset, enabling exogenous integration of soluble factors into an engineered hematopoietic stem cell for enhance maintenance potential of a quiescent stem cell population.

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