Early HSC engraftment kinetics at the single cell level 

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Is the HSC “bias”-really an HSC “bias”?

Biases in HSC output do not necessarily have to originate at the HSC level if one considers HSC differentiation as a multi-step process. The proliferation of a more committed progenitor subset could result in the observation of an HSC bias too. In other words one can ask, if heterogeneity in HSCs is necessary for peripheral blood clone size heterogeneity or if a neutral model can explain clone size differences (Xu, Kim, Chen, & Chou, 2018). Is the HSC bias really an inherent, stable phenotype of HSC clones? Several aspects are in line with a real “HSC bias”. Some data suggests kinetic phenomena and proliferation of more committed progenitors could play an important role too. In the next sections I will discuss HSC marker expression, the relation of HSC and mature cell clonality, and secondary transplantations with respect to this question.
Speaking for an inherent HSC bias is the fact that markers for the isolation of biased-HSCs have been described. These are for example CD229 (Carrelha et al., 2018)(Oguro et al., 2013), CD86 (Shimazu et al., 2012), CD41 (Gekas & Graf, 2013), BMP-activation (Crisan et al., 2015a), Vwf (Pinho et al., 2018)(Carrelha et al., 2018) (Sanjuan-Pla et al., 2013), Mfn2 (Luchsinger, de Almeida, Corrigan, Mumau, & Snoeck, 2016), Msi2 (Park et al., 2014), Hdc (Xiaowei Chen et al., 2017), CD61 (Mann et al., 2018), and LIGHT (Xufeng Chen et al., 2018), Per2 (J. Wang et al., 2016). Also, the LSK side population (SP) gating of HSCs based on Hoechst 33342 staining has been described to allow discrimination of differently biased HSCs. Myeloid-, and lymphoid-biased HSCs were described to lay in the lower and upper LSK SP gates respectively (Challen, Boles, Chambers, & Goodell, 2010).
The relationship of HSC and other committed progenitor clones, with mature cells based on which lineage biases are defined suggests kinetic phenomena could play a role in “bias” detection. In general, the percentage of HSC clones reported to be found back in mature cells is moderate after bulk transplantation. Repeatedly a number of HSC clones detected through cellular barcodes or VIS analysis have not been found back in mature hematopoietic cells (E. Verovskaya et al., 2013) (Evgenia Verovskaya, Broekhuis, et al., 2014) (Biasco et al., 2016) (Aiuti et al., 2013) (Lu et al., 2019); implying that not all HSC are active after bulk transplantation, but that some can be quiescent, senescent, or self-renewing without differentiation. The clonality of other progenitor compartments has in general been reported to be similar to mature cells (Lu et al., 2011) (Lu et al., 2019) (Biasco et al., 2016), meaning all clones differentiate (to some extent). Some studies allowed to relate the presence and abundance of clones in HSC and other progenitor compartments to lineage biases in the mature subsets. In these studies, both lymphoid and myeloid biased clones could be found back in HSCs which implies that both do have some self-renewal capacity. The percentage of balanced, myeloid-, and lymphoid-biased clones which is present in the HSC compartment was though not the same. In Sanggu Kim et al., the majority of the balanced (57-96%) and myeloid-biased clones (38-80%) was found back in the HSPC pool. In contrast only 17-50% of lymphoid-biased clones were found back (Sanggu Kim et al., 2014). Likewise, in Aiuti et al. the percentage of lymphoid VIS found back in bone marrow was much lower than for VIS found in myeloid cells (Aiuti et al., 2013). Also, in Carrelha et al., lymphoid-biased and lymphoid-restricted reconstitution, which was always proceeded by multilineage reconstitution, was not associated with long-term reconstitution of the HSC compartment. This could be in line with some lymphoid-biases being caused by the long lifespan of lymphoid cells and not reflecting the lymphoid bias of a currently active HSC. Alternatively, lymphoid-biased HSC might self-renew much less than myeloid-biased HSC and be present under the detection limit. To assess the change of biases over time can help to discriminate these two scenarios.
In the most thorough study monitoring biases over time these were found to be “extremely” stable (Koelle et al., 2017). This would again imply an inherent HSC bias. The comparison of bias stability upon repeated transplantation, is however rather contradicting this idea. Cells lying in the immunophenotypically defined HSC gate do not all lead to reconstitution upon secondary transplantation. This is the case for 86% of the myeloid-restricted progenitors identified by Yamamoto et al after single cell transplantation (lymphoid-restriction was not identified in this study) (Yamamoto et al., 2013b). When this was compared between differently biased HSCs it was repeatedly the lymphoid-biased HSCs which showed less reconstitution capacity upon secondary transplantation. In Dykstra et al. 90% of myeloid biased HSC were able to reconstitute upon secondary and third transplantation. None of the lymphoid-biased HSCs could (Dykstra et al., 2007). Likewise, In Grosselin et al., 5/13 of myeloid-biased but only 3/13 lymphoid-biased clones differentiated upon secondary transplantation. All of this could still be caused by a much lower self-renewal of lymphoid-biased HSC than myeloid-biased HSC; lymphoid-biased HSC might not be transplanted. A real hint towards kinetic phenomena underlying biases comes from the comparison of biases before and after secondary transplantation. Repeatedly > 50% of clones did keep their general lineage bias upon secondary transplantation (Dykstra et al., 2007) (Grosselin, Sii-Felice, Payen, Chretien, Roux, et al., 2013)(Carrelha et al., 2018). However, when a switch in output pattern was observed, this was always a switch from a platelet, to a myeloid, to a multilineage, to a lymphoid bias. In Grosselin et al. and Dykstra et al. clones switched for example sequentially from the a to the b, g , and d category. In Carrelha et al., where Mk- restricted HSC were transplanted, these either maintained high levels of Mk-restricted secondary reconstitution, or produced some low levels of erythroid, myeloid or even lymphoid cells (Carrelha et al., 2018). Also, in Verovskaya et al the number of lymphoid-restricted clones after secondary transplantation was higher (13/36) than after primary transplantation (6/43). Although here one single clone switched from lymphoid to myeloid cell production (Evgenia Verovskaya, Broekhuis, et al., 2014). In Morita et al., some cells displayed barely detectable myeloid engraftment in primary-recipient mice, but progressive and multilineage reconstitution in secondary recipient mice (the corresponding HSC were termed “latent”) (Morita et al., 2010). The few data available comparing in situ lineage biases to biases after transplantation describe both a change and stability of bias. In Rodriguez-Fraticelli et al., Mk-restricted HSC become multilineage-outcome HSC after transplantation (Rodriguez-Fraticelli et al., 2018). In Yu et al. stability of lineage biases in situ and after transplantation is seen, but only few clones are assessed and no statistics are given (V. W. C. Yu et al., 2016).
In recent years a high heterogeneity in the lineage output of HSC has been described. Notably Mk-, MkE-, MkEM-restricted, and lymphoid- and myeloid-biased HSC have been described after transplantation and also in vivo. Markers for lymphoid and myeloid-biased HSCs have been described implying bias to be a stable characteristic. However, individual HSC can also change bias and this has been described to follow a constant flow from erythroid, to myeloid and to lymphoid bias. Kinetic phenomena as production kinetics and life span of different hematopoietic lineages might therefore be one cause for the detection of lineage biases. The heterogeneity of HSC has profound implications for our understanding of hematopoiesis in steady state, under homeostatic challenges, during pathologies, and for clinical applications. Also our understanding of cytokine action on hematopoietic cells has been influenced by the discovery of HSC heterogeneity.

The effect of cytokines on HSC differentiation

HSCs guarantee the production of a particularly large number of different cell types with each having a crucial and specific role for the functioning of our body. When physiological needs change, following invasion of pathogens, malignancies, or mere physical activity, also the requirement for the production of each of these different hematopoietic cell types changes. The fast and adequate adaptation of the hematopoietic differentiation is needed to match the current demand. Hormones and cytokines, as erythropoietin, are important players in this process and can act on cells throughout the hematopoietic tree, including on HSCs.

Cytokines act throughout the hematopoietic tree

For a long time, the enhanced production of specific hematopoietic cell subsets in times of need was mainly considered to results from the induction of proliferation of mature hematopoietic cells, or uni-lineage committed hematopoietic progenitors. A prime example is the proliferation of mature antigen-specific lymphoid cells upon antigen-recognition to guarantee an adaptive immune response after pathogen exposure. Also during innate immune responses this principle was considered to be major. Emergency granulopoiesis, the rapid production of neutrophils after infection, has for example been described to be initiated by the cytokine-mediated induction of proliferation and maturation in committed myeloid progenitor cells (Manz & Boettcher, 2014; J. L. Zhao & Baltimore, 2015). Likewise, erythropoietin was described to induce erythroid cell production in anemic conditions by increasing survival of committed erythroid progenitors (Richmond, Chohan, & Barber, 2005). Only more recently, the action of cytokines on less committed hematopoietic progenitors gained more/new interest. To allow the production of a specific hematopoietic cell type, cytokines have been envisaged to act here, not by induction of proliferation, but rather through the instruction of lineage, meaning a “biasing” of differentiation. Such effect has for example been shown for M-CSF, and IFN-g acting on GMP (Rieger, Hoppe, Smejkal, Eitelhuber, & Schroeder, 2009) (De Bruin et al., 2012). More recently, also insulin has been described to induce such bias, namely a lymphoid lineage commitment in MPPs (Xia et al., 2015). All in all, it seems today established that the combination of cytokine-induced proliferation and lineage instruction can regulate hematopoietic differentiation for most hematopoietic progenitors. The action of cytokines on HSC themselves is still more controversial.

Cytokines and HSCs-what, where, and how?

That cytokines can act directly on HSC differentiation is obvious from in vitro culture. Still, the precise action and importance of different cytokine actions on HSCs during fast physiological responses, and especially for the production of specific hematopoietic cell subsets remains unclear. Different cytokines have been shown to regulate proliferation of HSCs in general. Luteinizing hormone (LH), for example, was described to limit HSC expansion of HSC during puberty via the LH-receptor (Peng et al., 2018). Contrary, IFN-a was described to promote exit of HSCs out of a dormant state into proliferation during infections, at least partly via the IFNa-R (Essers et al., 2009). Also for IFN-g such effect has been suggested (Baldridge, King, Boles, Weksberg, & Goodell, 2010).
Such regulation of proliferation of HSC in general, however can only lead to general increases or decreases of hematopoietic output. For the production of specific hematopoietic cell subsets, two different mechanisms of cytokine actions have been suggested: selective proliferation of “biased” HSC and, as already established for other hematopoietic progenitors, the direct instruction or “biasing” of HSC differentiation. Indeed, for both phenomena some first evidence exists. Two studies from the Challen group suggested cytokine-induced selective proliferation of HSC (Matatall, Shen, Challen, & King, 2014) (Challen et al., 2010). Matatall et al. suggested IFN-g would selectively promote the differentiation, and thereby depletion, of myeloid-biased HSCs defined based on SP distribution. They tracked down this effect to a 20-fold increased expression of IFN-g receptor 1 in myeloid-biased HSCs (Matatall et al., 2014). Likewise, Challen et al. described TGF-b as inducer of proliferation for myeloid-biased, but not lymphoid-biased HSCs (Challen et al., 2010). Two other studies suggested cytokine-induced lineage instruction on the HSC level (Mossadegh-Keller et al., 2013) (Grover et al., 2014). In a study by Mossadegh-Keller et al. the effect of M-CSF on HSC differentiation was assessed. It could be establish using video imaging that culture of HSCs with M-CSF increased the expression of myeloid genes including the myeloid master transcription factor PU.1. Also in vivo, high systemic levels of M-CSF directly stimulated M-CSF receptor-dependent PU.1 expression. Furthermore, the transplantation of in vivo M-CSF primed HSC in mice resulted in an increased ratio of GMP to MEP progenitors in spleen after 2 weeks and an increased myeloid to lymphoid cell ratio in peripheral blood after 4 weeks. All of this led the authors to conclude that M-CSF directly induced a myeloid lineage bias in single HSCs. A recent study could however not confirm parts of the study, namely the M-CSF induced expression of the PU.1 in HSC in vitro (Etzrodt et al., 2019). The effect of M-CSF on HSC stays thereby open. The second study suggesting cytokine-induced lineage instructions on the HSC level, studied the effect of EPO on HSCs (Grover et al., 2014) (see also next section). Grover et al. could observe that increased systemic EPO levels, changed the ratio of erythroid to other lineage-producing hematopoietic progenitors. Besides, in vitro exposure of HSC with EPO changed their transcriptome, increasing erythroid-, and decreasing the expression of myeloid-specific genes. Finally, transplantation of in vivo EPO-exposed HSCs in mice resulted in higher donor chimerism in erythroid cells than in myeloid cells in peripheral blood after 4 weeks. The authors concluded, that EPO induced an “erythropoietic superhighway” by skewing differentiation towards the erythroid lineage at every bifurcation in the hematopoietic tree starting from the HSC. As EPO was administered systemically in the study by Grover et al. it is however not clear if the effect of EPO on the differentiation of HSC was really direct, and linked to the transcriptional changes observed after in vitro culture with EPO.
Different mechanisms have been suggested by which cytokines can act on hematopoietic progenitors and HSCs, to increase the production of one specific hematopoietic lineage when needed. First evidence for different mechanisms has been given, but data is to date rather sparse and not fully conclusive. This is also the case for the cytokine EPO. The action of EPO on HSC and other (hematopoietic) cell subsets remains to be reconciled.

Erythropoietin and HSCs

Erythropoietin-classical view and new applications

Among all the different hematopoietic cells originating from HSCs, one cell type dominates in terms of numbers and turn-over. Of the 90% of our bodies cells comprised of hematopoietic cells, around 84% have been approximated to be erythrocytes (Sender et al., 2016). With an estimated 1% of daily renewal (Koury, 2016), erythropoiesis, the generation of erythrocytes from HSC, is thereby among the most productive hematopoietic processes. In the classical model of hematopoiesis, erythropoiesis proceeds from the MEP, to the generation of the restricted erythroid blast forming unit (BFU-E) and colony forming unit (CFU-E) progenitors, which further differentiate into pro-erythroblasts (ProEB), erythroblasts, and finally reticulocytes. These reticulocytes mature in the blood stream into the terminally differentiated erythrocytes which transport oxygen throughout the body (Koury & Haase, 2015). Considered as the major regulator of erythropoiesis is the cytokine erythropoietin (EPO), a 30 kDa, highly glycosylated protein, mainly expressed by a subset of adult renal peritubular interstitial cells. Although EPO is constantly secreted by these cells (with circadian rhythm) to guarantee the normal physiological erythroid turn-over, its production increases dramatically in hypoxic conditions (up to 1000 fold from the homeostatic serum concentrations of 2,6-18,5 mIU/ml (Mayo clinic) (Grote Beverborg et al., 2015) (Kaushansky, 2006)) to augment erythrocyte production. On a molecular level, this effect has been attributed to an EPO-induced increase in viability of CFU-E and ProEB. Both CFU-E and ProEB express the EPO receptor (EPOR) dimer in complex with other associated proteins as the transferrin receptor 1 and 2 (Tfr1/2), and Scribble at the highest recorded levels (1000 EPOR/cell)(Khalil et al., 2018) (Forejtnikova et al., 2010). Through binding of EPO to the EPOR complex, conformational changes in the receptor are thought to activate a cascade of signaling pathways from Janus kinase-2 over STAT5, Erk, PI3K-Akt, STAT3, which finally lead to an increased expression of anti-apoptotic proteins as Bcl2, and a decreased expression of apoptotic proteins as Fas and Fas ligand (Richmond et al., 2005). Thereby, EPO is believed to inhibit a local negative feedback loop based on Fas and Fas ligand expression of the erythroid progenitors at their site of production (Socolovsky, 2007). The potent action of EPO in increasing erythroid cell production, made recombinant EPO become an important medication. Indeed, recombinant EPO is today one of the most sold biopharmaceuticals in the world (Walsh, 2014) and different EPO formulations are routinely used in the clinics to counteract anemia in many different settings, including chronic renal failure, malignancy, prematurity, HIV infection, post-chemotherapy, post-radiotherapy, post-transplantation, or during surgeries when blood transfusion is disallowed.

Table of contents :

Chapter I: General introduction 
1.1 HSC definition and HSC differentiation models
1.2 Lineage tracing in the study of HSC differentiation
1.3 Lineage tracing of HSC at the single cell level by cellular barcoding
1.4 Lineage tracing reveals heterogeneity in HSC output-HSCs are “biased”
1.5 The effect of cytokines on HSC differentiation
1.6 Erythropoietin and HSCs
1.7 Aims of my thesis
Chapter 2: Early HSC engraftment kinetics at the single cell level 
2.1 Introduction
2.2 Results
2.3 Discussion
2.4 Materials and methods
2.5 Supplementary information
Chapter 3: The effect of erythropoietin on HSC differentiation 
3.1 Introduction
3.2 Results
3.3 Discussion
3.4 Materials and methods
3.5 Supplementary information
Chapter 4: What next? Outlook on cellular barcoding
4.1 The promises of available HSC barcoding libraries
4.2 HSC barcoding entering the “omics” era
4.3 HSC barcoding and other dimensions
4.4 Beyond the single-cell level, intra-clonal heterogeneity

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