Developments in Hematopoietic Stem Cell Expansion and Gene Editing Technologies.

Ex Vivo Expanded Hematopoietic Stem Cells Overcome the MHC Barrier in Allogeneic Transplantation

Advantages and challenges of ex vivo generation and expansion of human hematopoietic stem cells from pluripotent stem cells.

Ontogeny of Hematopoiesis

BackgroundThe aim of this study was the ex vivo expansion of Umbilical Cord Blood hematopoietic stem cells on biocompatible nanofiber scaffolds.Materials and MethodsCD133+ hematopoietic stem cells were separated from umbilical cord blood using MidiMacs (positive selection) system by means of monocolonal antibody CD133 (microbeads); subsequently, flowcytometry method was done to assess the purity of separated cells. Isolated cells were cultured on plate (2 Dimensional) and fibronectin conjugated polyethersulfon nanofiber scaffold, simultaneously (3 Dimensional). Colony assay test was performed to show colonization ability of expanded cells.ResultsCell count analysis revealed that expansion of hematopoietic stem cells in 2dimensional (2D) environment was greater than 3dimensional (3D) condition (p= 0.01). Assessment of stem cell- phenotype after expansions was performed by flowcytometric analysis which is showed that the maintenance of CD133 marker in expanded cells in 3 dimensional condition were higher than expanded cells in 2 dimensional condition (p=0.01). Moreover, colony assay test was performed before and after of expansion to show colonization ability of expanded cells both in 3D and 2D culture and results revealed more ability of 3D culture compared with 2D culture (p= 0.03).ConclusionThe results of current study confirmed that umbilical cord blood CD133+ haematopoietic stem cells are able to expand on fibronectin conjugated polyethersulfon scaffold. These findings indicated that 3D is a proper and valuable cell culture system for hematopoietic stem cells expansion, compared to 2D in invitro situation.

Pleiotrophin Is a Growth Factor for Hematopoietic Stem Cells and Induces Stem Cell Self-Renewal

Multi-Log Clonal Ex-Vivo Expansion of Long Term Lympho-Myeloid Hematopoietic Stem Cells by Nup98-Hox Fusion Genes.

Objectives: We aimed to identify the potential HOXB4/HOXC4 downstream effectors and elucidate their regulatory mechanism in the expansion of hematopoietic stem cell (HSC).Methods: The microarray data GSE24379 were downloaded from Gene Expression Omnibus database, including 12 human CD34+ hematopoietic cells with irradiated EGFP-, HOXB4-, or HOXC4-transduced MS-5 cells, respectively. Then common differentially expressed genes (DEGs) in HOXB4- and HOXC4-treated hematopoietic cells (HOXB4&HOXC4.DEGs) were screened out. Protein–protein interaction (PPI) network was constructed and functional modules analysis was performed. Pathway enrichment analysis was performed using the Database for Annotation Visualization and Integrated Discovery. Besides, transcription regulatory network (TRN) was constructed to screen transcription factors (TFs) corresponding to HOXB4&HOXC4.DEGs.Results: A total of 408 HOXB4&HOXC4.DEGs (373 up- and 35 down-regulated) in hematopoietic cells were identified. Tumor protein p53 (TP53) had the highest degrees in PPI network. Cyclin B1 (CCNB1) was a hub node in Cluster 1. V-myc avian myelocytomatosis viral oncogene homolog (MYC) and MYC-associated factor X (MAX) were important TFs with higher degrees. Meanwhile, MYC, TP53, and CCNB1 were significantly enriched in cell cycle.Conclusion: MYC, MAX, TP53, and CCNB1 may be crucial HOXB4/HOXC4 downstream molecules potentially involved in HSCs expansion, and HOXB4 and HOXC4 homeoprotein could display positive effects on expansion of human HSCs via regulating these genes.

<div>Abstract<p>Clonal hematopoiesis results from somatic mutations in cancer driver genes in hematopoietic stem cells. We sought to identify novel drivers of clonal expansion using an unbiased analysis of sequencing data from 84,683 persons and identified common mutations in the 5-methylcytosine reader <i>ZBTB33</i> as well as in <i>YLPM1, SRCAP</i>, and <i>ZNF318</i>. We also identified these mutations at low frequency in patients with myelodysplastic syndrome. <i>Zbtb33</i>-edited mouse hematopoietic stem and progenitor cells exhibited a competitive advantage <i>in vivo</i> and increased genome-wide intron retention. <i>ZBTB33</i> mutations potentially link DNA methylation and RNA splicing, the two most commonly mutated pathways in clonal hematopoiesis and myelodysplastic syndromes.</p>Significance:<p>Mutations in known driver genes can be found in only about half of individuals with clonal hematopoiesis. Here, we performed a somatic mutation discovery effort in nonmalignant blood samples, which identified novel candidate genes that may play biological roles in hematopoietic stem cell expansion and hematologic malignancies.</p><p><i>This article is highlighted in the In This Issue feature, p. 403</i></p></div>

<div>Abstract<p>Clonal hematopoiesis results from somatic mutations in cancer driver genes in hematopoietic stem cells. We sought to identify novel drivers of clonal expansion using an unbiased analysis of sequencing data from 84,683 persons and identified common mutations in the 5-methylcytosine reader <i>ZBTB33</i> as well as in <i>YLPM1, SRCAP</i>, and <i>ZNF318</i>. We also identified these mutations at low frequency in patients with myelodysplastic syndrome. <i>Zbtb33</i>-edited mouse hematopoietic stem and progenitor cells exhibited a competitive advantage <i>in vivo</i> and increased genome-wide intron retention. <i>ZBTB33</i> mutations potentially link DNA methylation and RNA splicing, the two most commonly mutated pathways in clonal hematopoiesis and myelodysplastic syndromes.</p>Significance:<p>Mutations in known driver genes can be found in only about half of individuals with clonal hematopoiesis. Here, we performed a somatic mutation discovery effort in nonmalignant blood samples, which identified novel candidate genes that may play biological roles in hematopoietic stem cell expansion and hematologic malignancies.</p><p><i>This article is highlighted in the In This Issue feature, p. 403</i></p></div>

Hematopoietic stem cells (HSC) are multi-potent cells that function to generate a lifelong supply of all blood cell types. During mammalian embryogenesis, sites of hematopoiesis change over the course of gestation: from extraembryonic yolk sac and placenta, to embryonic aorta-gonad-mesonephros region, fetal liver, and finally fetal bond marrow where HSC reside postnatally. These tissues provide microenviroments for de novo HSC formation, as well as HSC maturation and expansion. Within adult bone marrow, HSC self-renewal and differentiation are thought to be regulated by two major cellular components within their so-called niche: osteoblasts and vascular endothelial cells. This review focuses on HSC generation within, and migration to, different tissues during development, and also provides a summary of major regulatory factors provided by osteoblasts and vascular endothelial cells within the adult bone marrow niche.

Ex Vivo Expansion of Hematopoietic Stem and Progenitor Cells Using a Natural Product, Garcinol

Members of the homeobox (Hox) gene family are known to mediate expansion of hematopoietic stem cells (HSCs) and progenitors. The absence of oncogenic properties promoted HOXB4 as prime candidate in the quest to expand HSCs for clinical purposes. Despite its potential to expand HSCs, studies with mutant mice showed that Hoxb4 is not essential for HSC generation and function under physiological conditions. Expression studies and the existence of functional redundancy in particular between paralog Hox genes suggest that HOXA4 might have potent properties to expand HSCs. Here we measured the ability of HOXA4 to promote ex vivo expansion of HSCs and progenitors using retrovirus-mediated overexpression. Our results provide evidence that HOXA4-transduced HSCs and primitive progenitors expand in culture conditions and demonstrate that the potential of expanded HOXA4 HSCs to give rise to mature myeloid and lymphoid progeny in normal proportions remained intact. Interestingly, constitutive overexpression of HOXA4 resulted in an unbalanced expansion of lymphoid/myeloid progenitors in bone marrow chimeras favorable to B-cell progenitors responsive to interleukin-7. This expansion was specific for these progenitors and not for the more primitive Whitlock-Witte-initiating cells. These data indicate that early stages of B-cell development associated with proliferation are in particular sensitive to HOXA4. Thus, this study supports the potential use of HOXA4 to expand both HSCs and B-cell progenitor populations for therapeutic strategies.

MODULATION OF HAEMATOPOIETIC PROGENITOR DEVELOPMENT BY FLT-3 LIGAND

Differential Effects of Epigenetic Modifiers on the Expansion and Maintenance of Human Cord Blood Stem/Progenitor Cells

In Vitro Expansion of Human Hematopoietic Cells with Delayed but Sustained Multi-Lineage Repopulating Activity