Encapsulation and recovery of murine hematopoietic stem and progenitor cells in a thiol-crosslinked maleimide-functionalized gelatin hydrogel

Biomaterial platforms are an integral part of stem cell biomanufacturing protocols. The collective biophysical, biochemical, and cellular cues of the stem cell niche microenvironment play an important role in regulating stem cell fate decisions. Three-dimensional (3D) culture of stem cells within biomaterials provides a route to present biophysical and biochemical stimuli such as cell-matrix interactions and cell-cell interactions via secreted biomolecules. Herein, we describe a maleimide-functionalized gelatin (GelMAL) hydrogel that can be crosslinked via thiol-Michael addition click reaction for the encapsulation of sensitive stem cell populations. The maleimide functional units along the gelatin backbone enables gelation via the addition of a dithiol crosslinker without requiring external stimuli (e.g., UV light, photoinitiator), reducing reactive oxide species generation. Additionally, the versatility of crosslinker selection enables easy insertion of thiol-containing bioactive or bioinert motifs. Hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) were encapsulated in GelMAL, with mechanical properties tuned to mimic the in vivo bone marrow niche. We report insertion of a cleavable peptide crosslinker that can be degraded by the proteolytic action of SortaseA, a mammalian-inert enzyme. Notably, SortaseA exposure preserves stem cell surface markers, an essential metric of hematopoietic activity used in immunophenotyping. This novel GelMAL system enables a route to producing artificial stem cell niches with tunable biophysical properties with intrinsic cell-interaction motifs and orthogonal addition of bioactive crosslinks.

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Runx1 downregulates stem cell and megakaryocytic transcription programs that support niche interactions

Blood ◽

10.1182/blood-2015-09-668129 ◽

2016 ◽

Vol 127 (26) ◽

pp. 3369-3381 ◽

Cited By ~ 21

Author(s):

Kira Behrens ◽

Ioanna Triviai ◽

Maike Schwieger ◽

Nilgün Tekin ◽

Malik Alawi ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Cell Adhesion ◽

Cell Fate ◽

Bone Marrow Niche ◽

Cell Fate Decisions ◽

Multipotent Progenitors ◽

Key Points

Key Points Runx1 is a key determinant of megakaryocyte cell-fate decisions in multipotent progenitors. Runx1 downregulates cell-adhesion factors that promote residency of stem cells and megakaryocytes in their bone marrow niche.

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Wnt-inhibitory factor 1 dysregulation of the bone marrow niche exhausts hematopoietic stem cells

Blood ◽

10.1182/blood-2010-09-305664 ◽

2011 ◽

Vol 118 (9) ◽

pp. 2420-2429 ◽

Cited By ~ 47

Author(s):

Christoph Schaniel ◽

Dario Sirabella ◽

Jiajing Qiu ◽

Xiaohong Niu ◽

Ihor R. Lemischka ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Wnt Signaling ◽

Cell Fate ◽

Sonic Hedgehog ◽

Hematopoietic Stem ◽

Cell Fate Decisions ◽

Cell Niche ◽

Wnt Inhibitory Factor 1 ◽

Stem Cell Pool

Abstract The role of Wnt signaling in hematopoietic stem cell fate decisions remains controversial. We elected to dysregulate Wnt signaling from the perspective of the stem cell niche by expressing the pan Wnt inhibitor, Wnt inhibitory factor 1 (Wif1), specifically in osteoblasts. Here we report that osteoblastic Wif1 overexpression disrupts stem cell quiescence, leading to a loss of self-renewal potential. Primitive stem and progenitor populations were more proliferative and elevated in bone marrow and spleen, manifesting an impaired ability to maintain a self-renewing stem cell pool. Exhaustion of the stem cell pool was apparent only in the context of systemic stress by chemotherapy or transplantation of wild-type stem cells into irradiated Wif1 hosts. Paradoxically this is mediated, at least in part, by an autocrine induction of canonical Wnt signaling in stem cells on sequestration of Wnts in the environment. Additional signaling pathways are dysregulated in this model, primarily activated Sonic Hedgehog signaling in stem cells as a result of Wif1-induced osteoblastic expression of Sonic Hedgehog. We find that dysregulation of the stem cell niche by overexpression of an individual component impacts other unanticipated regulatory pathways in a combinatorial manner, ultimately disrupting niche mediated stem cell fate decisions.

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Development of an Inexpensive Raman-Compatible Substrate for the Construction of a Microarray Screening Platform

10.26434/chemrxiv.12455816 ◽

2020 ◽

Author(s):

Isamar Pastrana-Otero ◽

Sayani Majumdar ◽

Aidan E. Gilchrist ◽

Brittney L. Gorman ◽

Brendan A. C. Harley ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Fate ◽

Living Cells ◽

Raman Microspectroscopy ◽

Partial Least Square ◽

Hematopoietic Stem ◽

Stem Cell Fate ◽

Cell Fate Decisions ◽

Extrinsic Cues

Biomaterial microarrays are being developed to facilitate identifying the extrinsic cues that elicit stem cell fate decisions to self-renew, differentiate and remain quiescent. Raman microspectroscopy, often combined with multivariate analysis techniques such as partial least square-discriminant analysis (PLS-DA), could enable the non-invasive identification of stem cell fate decisions made in response to extrinsic cues presented at specific locations on these microarrays. Because existing biomaterial microarrays are not compatible with Raman microspectroscopy, here, we develop an inexpensive substrate that is compatible with both single-cell Raman spectroscopy and the chemistries that are often used for biomaterial microarray fabrication. Standard deposition techniques were used to fabricate a custom Raman-compatible substrate that supports microarray construction. We validated that spectra from living cells on functionalized polyacrylamide (PA) gels attached to the custom Raman-compatible substrate are comparable to spectra acquired from a more expensive commercially available substrate. We also showed that the spectra acquired from individual living cells on functionalized PA gels attached to our custom substrates were of sufficient quality to enable accurate identification of cell phenotypes using PLS-DA models of the cell spectra. We demonstrated this by using cells from laboratory lines (CHO and transfected CHO cells) as well as adult stem cells that were freshly isolated from mice (long-term and short-term hematopoietic stem cells). The custom Ramancompatible substrate reported herein may be used as an inexpensive substrate for constructing biomaterial microarrays that enable the use of Raman microspectroscopy to non-invasively identify the fate decisions of stem cells in response to extrinsic cues.

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Cell Cycle Regulation and Cell Fate Decisions in Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v106.11.1349.1349 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1349-1349

Author(s):

Emmanuelle Passegue ◽

Amy J. Wagers ◽

Sylvie Giuriato ◽

Wade C. Anderson ◽

Irving L. Weissman

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

Cell Fate ◽

Cell Cycle Regulation ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cell Fate Decisions ◽

Cycle Regulation

Abstract The blood is a perpetually renewing tissue seeded by a rare population of adult bone marrow hematopoietic stem cells (HSC). During steady-state hematopoiesis, the HSC population is relatively quiescent but constantly maintains a low numbers of cycling cells that differentiate to produce the various lineage of mature blood cells. However, in response to hematological stress, the entire HSC population can be recruited into cycle to self-renew and regenerate the blood-forming system. HSC proliferation is therefore highly adaptative and requires appropriate regulation of cell cycle progression to drive both differentiation-associated and self-renewal-associated proliferation, without depletion of the stem cell pool. Although the molecular events controlling HSC proliferation are still poorly understood, they are likely determined, at least in part, by regulated expression and/or function of components and regulators of the cell cycle machinery. Here, we demonstrate that the long-term self-renewing HSC (defined as Lin−/c-Kit+/Sca-1+/Thy1.1int/Flk2−) exists in two distinct states that are both equally important for their in vivo functions as stem cells: a numerically dominant quiescent state, which is critical for HSC function in hematopoietic reconstitution; and a proliferative state, which represents almost a fourth of this population and is essential for HSC functions in differentiation and self-renewal. We show that when HSC exit quiescence and enter G1 as a prelude to cell division, at least two critical events occur: first, during the G1 and subsequent S-G2/M phases, they temporarily lose efficient in vivo engraftment activity, while retaining in vitro differentiation potential; and second, they select the particular cell cycle proteins that are associated with specific developmental outcomes (self-renewal vs. differentiation) and developmental fates (myeloid vs. lymphoid). Together, these findings provide a direct link between HSC proliferation, cell cycle regulation and cell fate decisions that have critical implications for both the therapeutic use of HSC and the understanding of leukemic transformation.

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Mitochondrial Control of Stem Cell State and Fate: Lessons From Drosophila

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.606639 ◽

2021 ◽

Vol 9 ◽

Author(s):

Satish Kumar Tiwari ◽

Sudip Mandal

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Fate ◽

Cell Types ◽

Extrinsic Factors ◽

Cell Fate Decisions ◽

Cell State ◽

Resident Stem Cells ◽

Stem Cell State

Over the years, Drosophila has served as a wonderful genetically tractable model system to unravel various facets of tissue-resident stem cells in their microenvironment. Studies in different stem and progenitor cell types of Drosophila have led to the discovery of cell-intrinsic and extrinsic factors crucial for stem cell state and fate. Though initially touted as the ATP generating machines for carrying various cellular processes, it is now increasingly becoming clear that mitochondrial processes alone can override the cellular program of stem cells. The last few years have witnessed a surge in our understanding of mitochondria’s contribution to governing different stem cell properties in their subtissular niches in Drosophila. Through this review, we intend to sum up and highlight the outcome of these in vivo studies that implicate mitochondria as a central regulator of stem cell fate decisions; to find the commonalities and uniqueness associated with these regulatory mechanisms.

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Development of an Inexpensive Raman-Compatible Substrate for the Construction of a Microarray Screening Platform

10.26434/chemrxiv.12455816.v1 ◽

2020 ◽

Author(s):

Isamar Pastrana-Otero ◽

Sayani Majumdar ◽

Aidan E. Gilchrist ◽

Brittney L. Gorman ◽

Brendan A. C. Harley ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Fate ◽

Living Cells ◽

Raman Microspectroscopy ◽

Partial Least Square ◽

Hematopoietic Stem ◽

Stem Cell Fate ◽

Cell Fate Decisions ◽

Extrinsic Cues

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An RNA Interference Screen Identifies the Cell Fate Determinants Msi2 and Prox1 as Novel Regulators of Hematopoietic Stem Cell Self-Renewal.

Blood ◽

10.1182/blood.v114.22.394.394 ◽

2009 ◽

Vol 114 (22) ◽

pp. 394-394

Author(s):

Kristin J Hope ◽

Sonia Cellot ◽

Stephen Ting ◽

Guy Sauvageau

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Fate ◽

Self Renewal ◽

Hematopoietic Stem ◽

Control Shrna ◽

Fate Determinants ◽

Cell Fate Determinants

Abstract Abstract 394 Hematopoietic stem cells (HSC) can not yet be unambiguously prospectively identified, a fact which has made it difficult to determine whether a segregation of cell fate determinants underlies the asymmetric/symmetric self-renewal of these cells or whether deregulation of such determinants could contribute to the pathogenesis of hematopoietic malignancies by inducing constitutive symmetric self-renewal divisions. We have addressed these questions through a functional genetics approach taking advantage of systematic RNAi to evaluate the function of conserved polarity factors and cell fate determinants in HSCs. From a list of 72 of such factors identified in the literature, 30 murine homologues were chosen based on their differentially higher level of expression in HSC-enriched populations as measured by qRT-PCR. For each candidate we designed 3 unique short hairpin RNA (shRNA) encoding retroviral constructs also carrying EGFP for the purposes of following transduced cells. Primitive hematopoietic cells enriched for HSC were infected at high efficiency with the library in an arrayed 96-well format and their in vivo reconstituting potential was then evaluated through competitive repopulating unit assays. Genes for which shRNA vectors altered late transplant EGFP levels below or above thresholds as defined by a control shRNA to luciferase were considered as hits. Using this approach, we identified and comprehensively validated 4 genes, including the RNA binding protein Msi2, for which shRNA-mediated depletion dramatically impairs repopulation but does not induce cell death or a cell cycle block. Importantly, we show that the loss in the repopulating ability of these shRNA transduced cells is mediated at the stem cell level and is not due to progenitor or downstream cell toxicity or to any defect in the process of bone marrow homing. Subsequent expression profiling indicated that Msi2 is also upregulated in HOXB4-overexpressing symmetrically expanding HSC in line with our findings that it functions as a positive HSC regulator and further suggesting that it represents a potential novel HSC marker. As well as finding HSC agonists, the RNAi screen identified the homeodomain containing transcription factor Prox1 as a negative HSC regulator since its shRNA-mediated transcript loss consistently led to the dramatic in vivo accumulation of EGFP+ transduced cells. Grafts comprised of Prox1 shRNA-transduced cells did not exhibit any lineage skewing however, repeatedly contained an average of 10-fold more primitive Lin-Sca+CD150+48- cells as compared to non-transduced donor cells within the same recipient or to control shRNA-luciferase grafts indicating Prox1 knockdown leads to a significant in vivo expansion of phenotypic HSCs. Moreover, following a 7 day in vitro culture, cells infected with shRNAs to Prox1 were both morphologically and immunophenotypically more primitive than control cells and when transplanted at this time yielded a significantly enhanced engraftment level relative to control shRNAs (51+/-6% GFP vs 8+/-3% GFP). These results further suggest that Prox1 reduction by RNAi expands functional HSCs in vitro. Together these findings have identified conserved cell fate determinants as important and novel regulators of murine hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

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MT1-MMP Regulates Hematopoiesis Through HIF-Mediated Chemo-/Cytokine Release From the Bone Marrow Niche,

Blood ◽

10.1182/blood.v118.21.3409.3409 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3409-3409

Author(s):

Chiemi Nishida ◽

Kaori Kusubata ◽

Yoshihiko Tashiro ◽

Ismael Gritli ◽

Aki Sato ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Cell Fate ◽

Conflicts Of Interest ◽

Stem Cell Differentiation ◽

Cell Phenotype ◽

Bone Marrow Niche ◽

Matrix Remodeling ◽

Hematopoietic Stem

Abstract Abstract 3409 Stem cells reside in a physical niche, a particular microenvironment. The organization of cellular niches has been shown to play a key role in regulating normal stem cell differentiation, stem cell maintenance and regeneration. Various stem cell niches have been shown to be hypoxic, thereby maintaining the stem cell phenotype, e.g. for hematopoietic stem cells (HSCs) or cancer stem cells. The bone marrow (BM) niche is a rich reservoir for tissue-specific pluripotent HSCs. Proteases, such as matrix metalloproteinases (MMPs) can modulate stem cell fate due to their proteolytic or non-proteolytic functions (abilities). We have investigated the role of membrane-type1 matrix metalloproteinase (MT1-MMP), known for its role in pericellular matrix remodeling and cell migration, in hematopoiesis. MT1-MMP is highly expressed in HSCs and stromal cells. In MT1-MMP−/− mice, release of kit ligand (KitL), stromal cell derived factor-1 (SDF-1/CXCL12), erythropoietin (Epo) and interleukin-7 were impaired resulting in erythroid, myeloid and T and B lymphoid differentiation. Addition of exogenous rec. KitL and rec. SDF-1 restored hematopoiesis in vivo and in vitro. Further mechanistic studies revealed that MT1-MMP in a non-proteolytic manner activates the HIF-1 pathway, thereby inducing the transcription of the HIF-responsive genes KitL, SDF-1 and Epo. These results suggested MT1-MMP as a critical regulator of postnatal hematopoiesis, which as a modulator of the HIF pathway alters critical hematopoietic niche factors necessary for terminal differentiation and or migration. Thus, our results indicate that MT1-MMP as a key molecular link between hypoxia and the regulation of vital HSC niche factors. Disclosures: No relevant conflicts of interest to declare.

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Mitochondrial Activity Determines Hematopoietic Stem Cell Fate Decisions

Blood ◽

10.1182/blood.v124.21.4327.4327 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4327-4327

Author(s):

Nicola Vannini ◽

Mukul Girotra ◽

Olaia M. Naveiras ◽

Vasco Campos ◽

Evan Williams ◽

...

Keyword(s):

Stem Cell ◽

Cell Fate ◽

Hematopoietic Stem Cell ◽

Conflicts Of Interest ◽

Mitochondrial Activity ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cell Fate Decisions

Abstract A tight control of hematopoietic stem cell (HSC) quiescence, self-renewal and differentiation is crucial for lifelong blood production. The mechanisms behind this control are still poorly understood. Here we show that mitochondrial activity determines HSC fate decisions. A low mitochondrial membrane potential (Δψm) predicts long-term multi-lineage blood reconstitution capability, as we show for freshly isolated and in vitro-cultured HSCs. However, as in vivo both quiescent and cycling HSCs have comparable Δψm distributions, a low Δψm is not per se related to quiescence but is also found in dividing cells. Indeed, using divisional tracking, we demonstrate that daughter HSCs with a low Δψm maintain stemness, whereas daughter cells with high Δψm have undergone differentiation. Strikingly, lowering the Δψm by chemical uncoupling of the electron transport chain leads to HSC self-renewal under culture conditions that normally induce rapid differentiation. Taken together, these data show that mitochondrial activity and fate choice are causally related in HSCs, and provides a novel method for identifying HSC potential after in vitro culture. Disclosures No relevant conflicts of interest to declare.

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Bone marrow-on-a-chip: Emulating the human bone marrow

10.1101/469528 ◽

2018 ◽

Author(s):

Stefan Sieber ◽

Annika Winter ◽

Johanna Wachsmuth ◽

Rhiannon David ◽

Maria Stecklum ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Culture System ◽

3D Culture ◽

In Vitro Cultivation ◽

Bone Marrow Niche ◽

Hematopoietic Stem

AbstractMultipotent hematopoietic stem and progenitor cells HSPC reside in specialized stem cell niches within the bone marrow, that provide a suitable microenvironment for lifelong maintenance of the stem cells. Meaningful in vitro models recapitulating the in vivo stem cell niche biology can be employed for both basic research as well as for applied sciences and represent a powerful tool to reduce animal tests in preclinical studies. Recently we published the generation of an in vitro bone marrow niche model, capable of long-term cultivation of HSC based on an organ-on-a-chip platform. This study provides a detailed analysis of the 3D culture system including matrix environment analysis by SEM, transcriptome analysis and system intrinsic differentiation induction. Furthermore, the bone marrow on a chip model can serve to multiply and harvest HSPC, since repeated cell removal not compromised the functionality of the culture system. The prolongation of the culture time to 8 weeks demonstrate the capacity to apply the model in repeated drug testing experiments. The quality of the presented system is emphasized by the differentiation capacity of long-term cultivated HSPC in vitro and in vivo. Transplanted human HSPC migrated actively into the bone marrow of irradiated mice and contributed to the long-term reconstitution of the hematopoietic system after four and eight weeks of in vitro cultivation.The introduced system offers a multitude of possible applications to address a broad spectrum of questions regarding HSPC, the corresponding bone marrow niche biology, and pathological aberrations.

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