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Stem cells are special cells with the distinctive capability to self-renew, forming a new pool of undifferentiated stem cells. They are also able to differentiate into lineage-specific cell types that are specialized and matured. Thus, stem cells are considered as the building blocks of tissues and organs in which they reside. Among the many types of stem cells, hematopoietic stem cells (HSCs) are the most studied adult stem cells and are considered as a promising source of cells for applications in the clinical and basic sciences. Historically, research on HSCs was initiated in the 1940s, where in a groundbreaking experiment, intravenously injected bone marrow (BM) cells prevented the death of irradiated mice by restoring blood cell production. Since then, HSCs have been studied and utilized in medical therapies and research for over several decades. Over time, more sophisticated tools have been developed to evaluate the behaviour of specifically purified subsets of hematopoietic cells that have the capacity to produce blood cells. One of the established tools is the colony-forming units (CFUs) assay. This assay facilitates the identification, enumeration, and analysis of colonies formed by differentiated hematopoietic stem and progenitor cells (HSPCs) from myeloid, erythroid and lymphoid lineages. Hence, the CFUs assay is a fundamental in vitro platform that allows functional studies on the lineage potential of an individual HSPCs. The outcomes of such studies are crucial in providing critical insights into hematopoiesis. In this review, we explore the fundamental discoveries concerning the CFUs assay by covering the following aspects: (i) the historical overview of the CFUs assay for the study of clonal hematopoiesis involving multilineage potential of HSPCs, (ii) its use in various experimental models comprising humans, mice/rodents, zebrafish and induced pluripotent stem cells (iPSCs) and (iii) research gaps and future direction concerning the role of CFUs assay in clinical and basic sciences. Overall, the CFUs assay confers a transformative platform for a better understanding of HSPCs biology in governing hematopoiesis.

Mesenchymal stroma cells (MSCs) have a high potential for novel cell therapy approaches in clinical transplantation. Commonly used bone marrow-derived MSCs (BM-MSCs), however, have a restricted proliferative capacity and cultures are difficult to standardize. Recently developed human embryonic stem cell-derived mesenchymal stroma cells (hES-MSCs) might represent an alternative and unlimited source of hMSCs. We therefore compared human ES-cell-derived MSCs (hES-MP002.5 cells) to normal human bone marrow-derived MSCs (BM-MSCs). hES-MP002.5 cells had lower yet reasonable CFU-F capacity compared with BM-MSC (8±3 versus 29±13 CFU-F per 100 cells). Both cell types showed similar immunophenotypic properties, i.e. cells were positive for CD105, CD73, CD166, HLA-ABC, CD44, CD146, CD90, and negative for CD45, CD34, CD14, CD31, CD117, CD19, CD 271, SSEA-4 and HLA-DR. hES-MP002.5 cells, like BM-MSCs, could be differentiated into adipocytes, osteoblasts and chondrocytes in vitro. Neither hES-MP002.5 cells nor BM-MSCs homed to the bone marrow of immune-deficient NSG mice following intravenous transplantation, whereas intra-femoral transplantation into NSG mice resulted in engraftment for both cell types. In vitro long-term culture-initiating cell assays and in vivo co-transplantation experiments with cord blood CD34+ hematopoietic cells demonstrated furthermore that hES-MP002.5 cells, like BM-MSCs, possess potent stroma support function. In contrast to BM-MSCs, however, hES-MP002.5 cells showed no or only little activity in mixed lymphocyte cultures and phytohemagglutinin (PHA) lymphocyte stimulation assays. In summary, ES-cell derived MSCs might be an attractive unlimited source for stroma transplantation approaches without suppressing immune function.

Erythropoiesis maintains a stable hematocrit and tissue oxygenation in the basal state, while mounting a stress response that accelerates red cell production in anemia, blood loss or high altitude. Thus, tissue hypoxia increases secretion of the hormone erythropoietin (Epo), stimulating an increase in erythroid progenitors and erythropoietic rate. Several cell divisions must elapse, however, before Epo-responsive progenitors mature into red cells. This inherent delay is expected to reduce the stability of erythropoiesis and to slow its response to stress. Here we identify a mechanism that helps to offset these effects. We recently showed that splenic early erythroblasts, 'EryA', negatively regulate their own survival by co-expressing the death receptor Fas, and its ligand, FasL. Here we studied mice mutant for either Fas or FasL, bred onto an immune-deficient background, in order to avoid an autoimmune syndrome associated with Fas deficiency. Mutant mice had a higher hematocrit, lower serum Epo, and an increased number of splenic erythroid progenitors, suggesting that Fas negatively regulates erythropoiesis at the level of the whole animal. In addition, Fas-mediated autoregulation stabilizes the size of the splenic early erythroblast pool, since mutant mice had a significantly more variable EryA pool than matched control mice. Unexpectedly, in spite of the loss of a negative regulator, the expansion of EryA and ProE progenitors in response to high Epo in vivo, as well as the increase in erythropoietic rate in mice injected with Epo or placed in a hypoxic environment, lagged significantly in the mutant mice. This suggests that Fas-mediated autoregulation accelerates the erythropoietic response to stress. Therefore, Fas-mediated negative autoregulation within splenic erythropoietic tissue optimizes key dynamic features in the operation of the erythropoietic network as a whole, helping to maintain erythroid homeostasis in the basal state, while accelerating the stress response.

New antifungal drugs are urgently needed due to the currently limited selection, the emergence of drug resistance, and the toxicity of several commonly used drugs. To identify drug leads, we screened small molecules using a Saccharomyces cerevisiae reporter bioassay in which S. cerevisiae heterologously expresses Hik1, a group III hybrid histidine kinase (HHK) from Magnaporthe grisea. Group III HHKs are integral in fungal cell physiology, and highly conserved throughout this kingdom; they are absent in mammals, making them an attractive drug target. Our screen identified compounds 13 and 33, which showed robust activity against numerous fungal genera including Candida spp., Cryptococcus spp. and molds such as Aspergillus fumigatus and Rhizopus oryzae. Drug-resistant Candida albicans from patients were also highly susceptible to compounds 13 and 33. While the compounds do not act directly on HHKs, microarray analysis showed that compound 13 induced transcripts associated with oxidative stress, and compound 33, transcripts linked with heavy metal stress. Both compounds were highly active against C. albicans biofilm, in vitro and in vivo, and exerted synergy with fluconazole, which was inactive alone. Thus, we identified potent, broad-spectrum antifungal drug leads from a small molecule screen using a high-throughput, S. cerevisiae reporter bioassay.

Human bone marrow contains bone progenitor cells that arise from multipotent mesenchymal stem cells. Seeding bone progenitor cells onto a scaffold can produce a 3D living composite with significant mechanical and biological potential. This article details laboratory and clinical findings from two clinical cases, where different proximal femoral conditions were treated using impacted allograft augmented with marrow-derived autogenous progenitor cells. Autologous bone marrow was seeded onto highly washed morselized allograft and impacted. Samples of the impacted graft were also taken for analysis. Both patients made an uncomplicated clinical recovery. Imaging confirmed defect filling with encouraging initial graft incorporation. Histochemical and alkaline phosphatase staining demonstrated that a live composite graft with osteogenic activity had been introduced into the defects. These studies demonstrate that marrow-derived cells can adhere to highly washed morselized allograft, survive the impaction process and proliferate with an osteoblastic phenotype, thus creating a living composite.