In a study published today in the journal Nature, the team reports that activating the protein causes blood stem cells to self-renew at least twelvefold in laboratory conditions.
Multiplying blood stem cells in conditions outside the human body could greatly improve treatment options for blood cancers like leukemia and for many inherited blood diseases.
Dr. Hanna Mikkola, a member of the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA and senior author of the study, has studied blood stem cells for more than 20 years.
“Although we’ve learned a lot about the biology of these cells over the years, one key challenge has remained: making human blood stem cells self-renew in the lab,” she said.
“We have to overcome this obstacle to move the field forward.”
Blood stem cells, also known as hematopoietic stem cells, are found in the bone marrow, where they self-renew as well as differentiate to create all types of blood cells.
Bone marrow transplants have been used for decades to treat people with some diseases of the blood or immune system.
However, bone marrow transplants have significant limitations: Finding a compatible bone marrow donor is not always possible, the patient’s immune system may reject the foreign cells, and the number of transplanted stem cells may not be enough to successfully treat the disease.
When blood stem cells are removed from the bone marrow and placed in laboratory dishes, they quickly lose their ability to self-renew, and they either die or differentiate into other blood cell types.
Mikkola’s goal, making blood stem cells self-renew in controlled laboratory conditions, would open up a host of new possibilities for treating many blood disorders—among them safer genetic engineering of patients’ own blood stem cells.
It could also enable scientists to produce blood stem cells from pluripotent stem cells, which have the potential to create any cell type in the body.
To uncover what makes blood stem cells self-renew in a lab, the researchers analyzed the genes that turn off as human blood stem cells lose their ability to self-renew, noting which genes turned off when blood stem cells differentiate into specific blood cells such as white or red cells.
They then put the blood stem cells into laboratory dishes and observed which genes shut down.
Using pluripotent stem cells, they made blood stem cell-like cells that lacked the ability to self-renew and monitored which genes were not activated.
They found that the expression of a gene called MLLT3 was closely correlated with blood stem cells’ potential to self-renew and that the protein generated by the MLLT3 gene provides blood stem cells with the instructions necessary to maintain its ability to self-renew.
It does this by working with other regulatory proteins to keep important parts of the blood stem cell’s machinery operational as the cells divide.
The researchers wondered if maintaining the level of the MLLT3 protein in blood stem cells in lab dishes would be sufficient to improve their self-renewing abilities.
Using a viral vector – a specially modified virus that can carry genetic information to a cell’s nucleus without causing a disease -the team inserted an active MLLT3 gene into blood stem cells and observed that functional blood stem cells were able to multiply in number at least twelvefold in lab dishes.
“If we think about the amount of blood stem cells needed to treat a patient, that’s a significant number,” said Mikkola, who is also a professor of molecular, cell and developmental biology in the UCLA College and a member of the UCLA Jonsson Comprehensive Cancer Center. “But we’re not just focusing on quantity; we also need to ensure that the lab-created blood stem cells can continue to function properly by making all blood cell types when transplanted.”
Other recent studies have identified small molecules – organic compounds that are often used to create pharmaceutical drugs – that help to multiply human blood stem cells in the laboratory.
When Mikkola’s team used the small molecules, they observed that blood stem cell self-renewal improved in general, but the cells could not maintain proper MLLT3 levels, and they also did not function as well when transplanted into mice.
“The previous discoveries with the small molecules are very important, and we’re building on them,” said Vincenzo Calvanese, a UCLA project scientist and the study’s co-corresponding author.
“Our method, which exposes blood stem cells to the small molecules and also inserts an active MLLT3 gene, created blood stem cells that integrated well into mouse bone marrow, efficiently produced all blood cell types and maintained their self-renewing ability.”
Importantly, MLLT3 made the blood stem cells self-renew at a safe rate; they didn’t acquire any dangerous characteristics such as multiplying too much or mutating and producing abnormal cells that could lead to leukemia.
The next steps for the researchers include determining what proteins and elements within blood stem cell DNA influence the on-off switch for MLLT3, and how this could be controlled using ingredients in the lab dishes. With that information, they could potentially find ways to switch MLLT3 on and off without the use of a viral vector, which would be safer for use in a clinical setting.
Stem cells exist at the apex of tissue development and can orchestrate embryonic differentiation of various tissues in an adult organism and regulate tissue homeostasis and regeneration after injury. Embryonic stem cells (ESCs) are the most undifferentiated stem cells and are capable of generating all cell types within the organism, whereas somatic tissue‐specific stem cells (e.g., hematopoietic stem cells, HSCs) can only regenerate cells within the same tissue.
In bone marrow, HSCs can produce blood cells on demand during homeostatic and regenerative hematopoiesis, and this capacity to produce the blood while maintaining the pool of HSCs is controlled by a delicate balance between self‐renewing and differentiating cell divisions. Self‐renewal is a specialized and highly regulated cell division producing one or two daughter cells with the same stem cell features as the parental stem cell.
This multifaceted mechanism has been the subject of extensive research because of clear implications in tissue homeostasis, regenerative medicine, and cancer therapy.
Leukemia is a cancer of the blood cells affecting either lymphoid or myeloid lineages that is caused by genetic and epigenetic alterations occurring in HSC or hematopoietic progenitor cell (HPC); the generated population of leukemic cells bear stem cell properties ensuring self‐preservation through their self‐renewal capacity while continuously feeding the neoplasm by differentiating into the bulk of leukemia cells. As a critical process regulating stem cell fate in normal and malignant hematopoiesis, self‐renewal is controlled by the specialized microenvironment, or niche, and intrinsic factors that guide the decision of a stem cell to either self‐renew or undergo differentiation, depending on the demand of the specific tissue.
The Krüppel‐like factor (KLF) family of proteins encompasses 17 zinc‐finger transcription factors, of which at least 5 have been implicated in key stem cell functions, such as self‐renewal 1, 2, 3, 4, 5, pluripotency 1, 2, 3, 5, embryogenesis 6, and erythropoiesis 7. Like other members of the family, KLF4 contains activation and repression domains that mediate recruitment of coactivators or corepressors and three zinc fingers that bind to guanine‐cytosine‐rich sequences such as CACCC found in gene regulatory promoters and enhancers 8, 9.
KLF4 has been increasingly studied since the landmark work describing its role in the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs), and the contribution of KLF4 to this process suggests a potential function in the preservation of stemness in other tissue stem cells 10.
KLF4 is expressed in a wide range of mammalian tissues and regulates diverse cellular processes during normal tissue homeostasis, including proliferation, survival, and differentiation.
In fact, KLF4 regulates self‐renewal in stem cells from different tissues (e.g., embryonal, intestine, and skin) in both homeostasis and cancer 1, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23. Although KLF4 has been studied more extensively in ESCs and solid tumors, there is emerging evidence of KLF4 involvement in the process of blood formation by regulating normal hematopoiesis and leukemia stem cells (LSCs).Go to:
Regulation of Self‐Renewal by KLFs in Pluripotent Stem Cells
Because most of what is known on the regulation of self‐renewal by KLFs has been described in ESCs, we will briefly review the main contributions before discussing the role of KLF4 in normal and malignant HSCs. ESCs are derived from the inner cell mass of the blastocyst—an early stage of the preimplantation embryo—and characterized for their capacity for self‐renewal and pluripotent differentiation of ESCs into any tissue and cell type 24, 25. ESCs regulate self‐renewal and pluripotency properties through cell‐intrinsic (NANOG, OCT4, SOX2, and KLFs) and ‐extrinsic factors (leukemia inhibitory factor, LIF) 1, 26, 27, 28, 29, 30, 31, 32, 33, 34. OCT4, SOX2, and NANOG were originally identified as members of a core regulatory pathway involved in the preservation of stemness by promoting self‐renewal and preserving pluripotency. Several KLFs (KLF1, KLF2, KLF4, KLF5, and KLF17) regulate self‐renewal in the embryos, ESCs, hematopoietic cells, and bone marrow stromal cells in mice, humans, and zebrafish (Table (Table1).1).
Among these KLFs, the most extensively studied and characterized are KLF2, KLF4, and KLF5, as they form part of a transcriptional circuitry that promotes self‐renewal in ESCs by activating pluripotency‐associated genes such as NANOG that inhibit their differentiation into primitive endoderm 1, 2. Abrogation of self‐renewal and terminal differentiation of ESCs by simultaneous knockdown of KLF2, KLF4, and KLF5 suggested a KLF regulatory circuitry, which was rescued by the introduction of RNAi‐resistant cDNA encoding these three factors 1.
The KLF2/KLF4/KLF5 triad controls self‐renewal by regulating the expression of genes involved in self‐renewal (Oct4, Sox2, Myc, and Tcl1) and pluripotency (Nanog, Esrrb, and Oct4), facilitating the formation of autoregulatory loops among Oct4, Sox2, Nanog, and Sall4 in ESCs (Fig. (Fig.3)3) 1, 2. Although most KLF proteins (KLF1–KLF10) can bind to the regulatory regions of Nanog, only KLF2, KLF4, and KLF5 are able to maintain murine ESCs in an undifferentiated state in the absence of LIF 2. Based on chromatin immunoprecipitation and sequencing analysis, KLF4 and KLF5 inhibit differentiation of mesoderm and endoderm in ESCs by activating targets other than NANOG 18.
These findings suggest that KLF2, KLF4, and KLF5 have overlapping functions but also exert distinct roles in self‐renewal of ESCs. Although KLF4 restores loss of stemness caused by deletion of Klf5 in murine ESCs 30, the fact that Klf4‐null embryos can develop to term suggests either that KLF4 is dispensable for embryogenesis or there is a functional compensation by other KLF proteins 1, 35.
KLF4 and NANOG are among the first transcription factors to shut down their transcription when ESCs exit pluripotency, and nuclear export of KLF4 upon ERK activation is a critical first step to exit the naive pluripotent state and initiate ESC differentiation 16, 36.
In addition, KLF4 is required for expression of the telomerase reverse transcriptase (TERT) in human ESCs and binds β‐catenin through protein‐to‐protein interaction, allowing the recruitment of this dimer to the Tert promoter in murine ESCs 17, 37. Finally, KLF4 acts as a fast mediator of LIF signaling through the activation of STAT3 to cooperate with OCT4 and SOX2 in activating the expression of NANOG while repressing the GATA6 and SOX17 genes, which are involved in endoderm differentiation 31.
In summary, several KLF transcription factors regulate key processes of stem cell function in ESCs, among which KLF2, KLF4, and KLF5 play prominent roles. The formation of a KLF circuitry may be exclusive to ESCs, as this mechanism has not been described in other stem cells.
Table 1
Roles of KLF in stem cell self‐renewal
| KLF proteins | Host organism | Tissue/cell analyzed | Key functionality | Genes regulated by KLFs |
|---|---|---|---|---|
| KLF1 | Mouse | Embryo 7 | Required for erythropoiesis 7 | Myc 7 |
| KLF2 | Mouse | Embryo and embryonic stem cells 1, 2, 7 | Promotes self‐renewal and pluripotency 1, 2 Required for erythropoiesis 7 | Nanog and Esrrb 1, 2 Myc 1, 7 Oct4, Tcl1, Nr5a2, Tbx3, Rif1, Sox2, Tcf3, Mycn, and Foxd3 1 Stella 2 |
| Human | Bone marrow stromal cells 3 | Promotes self‐renewal and pluripotency 3 | Oct4, Nanog, and Rex1 3 | |
| Zebrafish | Embryo 6 | Required for embryogenesis 6 | Oct4 6 | |
| KLF4 | Mouse | Embryonic stem cells 1, 2 | Promotes self‐renewal and pluripotency 1, 2 | Nanog and Esrrb 1, 2 Tcl1, Myc, Nr5a2, Tbx3, Nanog, Esrrb, Rif1, Oct4, Sox2, Tcf3, Mycn, and Foxd3 1 Stella 2 |
| Hematopoietic cells 4 | Promotes self‐renewal 4 | |||
| KLF5 | Mouse | Embryonic stem cells 1, 2, 5 | Promotes self‐renewal and pluripotency 1, 2, 5 | Nanog 1, 2, 5 Esrrb 1, 2 Oct4 and Sox2 1, 5 Tcl1, Myc, Nr5a2, Tbx3, Esrrb, Rif1, Oct4, Tcf3, Mycn, and Foxd3 1 Stella 2 |
| KLF17 | Zebrafish | Embryo 6 | Required for embryogenesis 6 | Oct4 6 |
Abbreviation: KLF, Krüppel‐like factor.
KLF4 regulates self‐renewal in ESCs and T‐ALL LICs. A KLF self‐regulated triad regulates self‐renewal in ESCs. In T‐ALL LICs, KLF4’s repression of the kinase MAP2K7 is prevented by CpG methylation of the KLF4 promoter. Abbreviations: ESCs, embryonic stem cells; JIP, JNK‐interacting protein; KLF, Krüppel‐like factor; LIC, leukemia‐initiating cell; LIF, leukemia inhibitory factor; Notch1‐ic, Notch1 intracellular; MAP2K7, mitogen‐activated protein kinase kinase 7; MAP3K, mitogen‐activated protein kinase 3; T‐ALL, T‐cell acute lymphoblastic leukemia.
Role of KLF4 in Normal HSCs
Regulation of HSC Self‐Renewal
The identification of mechanisms that promote ex vivo self‐renewing expansion is the Holy Grail in HSC research and is pursued by many groups aiming at bone marrow transplant and cell and gene therapy applications.
The bone marrow milieu modulates stemness at different levels through secreted factors (stem cell factor, thrombopoietin, interleukin‐3 [IL3], IL‐6, IL‐11, and fms‐like tyrosine kinase 3 [FLT3]), inflammatory cytokines (e.g., tumor necrosis factor alpha and interferon gamma), hypoxia, the extracellular matrix, and topographic direction of the mitotic spindle with respect to cellular components of the niche during cell division, which could lead to losses of key cellular interactions and an asymmetric distribution of intracellular components.
This specialized milieu delivers signals to HSCs through factors recognized by the corresponding receptors that translate information to nuclei, where transcription factors regulate the expression of genes involved in the control of self‐renewal. Some of the extrinsic mechanisms regulating HSCs are NOTCH1, hedgehog, WNT, EP receptor for prostaglandin E2, angiopoietin‐like protein 5, and pleiotrophin (review and references therein 38, 39, 40, 41) (Fig. (Fig.1).1).
It is not clear whether KLF4 plays a role in the regulation of these extrinsic mechanisms (Fig. (Fig.1),1), although KLF4 can inhibit the WNT pathway in intestinal epithelium through interaction with β‐catenin and repress the expression of NOTCH1 in keratinocytes, whereas NOTCH1 inhibits the expression of KLF4 in intestinal epithelium 42, 43, 44.
Self‐renewal is regulated by extrinsic (stem cell niche in bone marrow) and intrinsic factors in HSCs. A potential regulatory role of KLF4 is indicated based on reports in other cell types. Abbreviations: ANGPTL‐5, angiopoietin‐like protein 5; FGF, fibroblast growth factor; HH, hedgehog; HSC, hematopoietic stem cells; KLF, Krüppel‐like factor; PGE2, prostaglandin E2.
In addition to the regulation by stem cell niches, many intrinsic factors have been described as positive regulators of HSC self‐renewal (e.g., HOXB4, RUNX1, BMI1, p53, miR‐126, FLT3, STAT5A, HMGA2, and SALL4; Fig. Fig.1)1) 45, 46, 47, 48, 49, 50, 51, 52. Collectively, intrinsic factors regulate cell fate during division at different levels, such as control of gene expression via transcriptional regulation (e.g., RUNX1 and STAT5A), metabolic sensing of nutrients and growth factors (e.g., mTORC1), response to hypoxia and metabolism (e.g., hypoxia‐inducible factor 1α), and development and aging (e.g., BMI1 and p16).
Reflecting the complexity of self‐renewal regulation, many of these factors have interconnected functions; for example, TCF7 regulates the expression of RUNX1 independently of WNT signaling, and the histone H2A deubiquitinase MYSM1 drives the recruitment of RUNX1 into the GFI1 locus, another transcription factor involved in HSC self‐renewal 53, 54, 55.
Although KLF4 has not been directly associated with the intrinsic regulation of HSC self‐renewal, a few reports suggest a potential role in steady‐state hematopoiesis, such as the inhibitory effect on BMI1 in intestinal cells, inhibition of mTORC1 during somatic cell reprogramming, and regulation of KLF4 by p53 in acute myeloid leukemia (AML) 56, 57, 58.
In addition to transcriptional regulation, the expression of genes involved in self‐renewal can be mediated through epigenetic mechanisms such as CpG methylation and histone modifications.
For example, mutations in DNMT3A lead to an increase in self‐renewal and upregulation of stemness genes in HSCs, and loss of DNMT3A promotes expansion of HSCs in the bone marrow 59, 60, 61. Interestingly, DNMT3A binds to the CpG island in the KLF4 promoter in endothelial cells, inducing DNA methylation and subsequent gene repression 62.
In addition to DNMT3A, IDH1/2 and TET2 mutations, often found in hematologic malignancies, can deregulate the pattern of genomic DNA methylation and aberrantly increase self‐renewal 63.
It was recently reported that TET2 binds to KLF4 through protein‐to‐protein interaction to drive locus‐specific demethylation during reprogramming of B cells into iPSCs 64.
Lastly, maintenance of telomere length through telomerase activity also plays a critical role in self‐renewal, as loss of telomerase results in reduced self‐renewal capacity of HSCs, as evaluated by serial transplantation, in addition to promoting carcinogenesis via genomic instability 65. Interestingly, KLF4 activates TERT expression through interaction with β‐catenin in ESCs 37, 66. Finally, factors involved in the differentiation toward different lineages, not listed here, could be considered negative regulators of self‐renewal because the alternative fate during cell division is differentiation.
KLF4 Regulates Self‐Renewal in Adult HSCs
In the hematopoietic system, KLF4 promotes macrophage and monocyte differentiation, macrophage polarization, survival of natural killer cells, secondary antibody responses in memory B cells, and dendritic cell development, whereas KLF4 inhibits homeostatic proliferation of naïve T cells 4, 67, 68, 69, 70, 71, 72, 73, 74.
The enrichment of KLF4 transcripts in human HSCs (CD34+ CD38lo Linlo) compared with HPCs (CD34+ CD38hi Linhi) led to the study of KLF4 in HSCs from fetal livers, because embryonic homozygous deletion results in postnatal lethality 35, 70, 75. Although clonogenic and competitive transplantation assays of fetal Klf4‐null HSCs showed normal colony formation in methylcellulose cultures and hematologic reconstitution of cytoablated recipient mice 70, the role of KLF4 in adult bone marrow HSCs has not been investigated.
Further supporting a potential role of KLF4 in adult HSCs, loss of the cell fate determinant lethal giant larvae homolog 1 increases self‐renewal, resulting in elevated numbers of HSCs and a competitive advantage after transplantation that is associated with KLF4 repression 41.
This finding suggests that inactivation of KLF4 might contribute to the regulation of self‐renewal in adult HSC and warrants the study of KLF4 using somatic gene deletion.
More information: Vincenzo Calvanese et al. MLLT3 governs human haematopoietic stem-cell self-renewal and engraftment, Nature (2019). DOI: 10.1038/s41586-019-1790-2
