Multiple genetically engineered humanized microenvironments in a single mouse
© The Author(s). 2016
Received: 10 February 2016
Accepted: 13 June 2016
Published: 28 June 2016
Immunodeficient mouse models that accept human cell and tissue grafts can contribute greater knowledge to human stem cell research. In this technical report, we used biomaterial implants seeded with genetically engineered stromal cells to create several unique microenvironments in a single mouse. The scope of study was focused on human CD34 hematopoietic stem/progenitor cell (HSPC) engraftment and differentiation within the engineered microenvironment.
A mouse model system was created using subdermal implant sites that overexpressed a specific human cytokines (Vascular Endothelial Growth Factor A (hVEGFa), Stromal Derived Factor 1 Alpha (hSDF1a), or Tumor Necrosis Factor Alpha (hTNFa)) by stromal cells in a three-dimensional biomaterial matrix. The systemic exposure of locally overexpressed cytokines was minimized by controlling the growth of stromal cells, which led to autonomous local, concentrated sites in a single mouse for study. This biomaterial implant approach allowed for the local analysis of each cytokine on hematopoietic stem cell recruitment, engraftment and differentiation in four different tissue microenvironments in the same host. The engineered factors were validated to have bioactive effects on human CD34+ hematopoietic progenitor cell differentiation.
This model system can serve as a new platform for the study of multiple human proteins and their local effects on hematopoietic cell biology for in vivo validation studies.
Immunodeficient mice lack immune cell activity and do not acutely reject xenogenic cells and tissues [1, 2]. These mouse strains have been an enabling tool for understanding human cell growth and functions in the context of living systems [3–6]. In particular, these mouse models have greatly advanced functional characterization of human hematopoietic stem and progenitor cells (HSPCs) [7, 8]. Human HSPCs can be intravenously injected and engrafted into a host immunodeficient mouse to form a chimeric hematopoietic system. These experiments have helped to identify and validate factors that control human HSPCs migration, engraftment, self-renewal, and differentiation in mice for mechanistic and therapeutic purposes . Yet, there still remain critical areas to improve these immunodeficient mouse models to better model human HSPCs . For example, human HSPCs do not engraft into a human stromal cell bed which may be important for long-term support of human stem cell function by direct physical contact or the local secretion of stromal soluble factors.
Several approaches have been tested in order to improve the functional support of human HSPCs in immunodeficient mice. First, human cytokines have been directly injected to a host mouse before and after human HSPCs injection that promoted engraftment and differentiation of human cells when compared to the control [11, 12]. Since single injection of human cytokines only produces a transient effect, repeated injections are typically required, which is cost-prohibitive and burdensome. Alternatively, human gene encoded lentiviral vectors  or plasmid deoxyribonucleic acids (DNAs)  have been applied to directly transfect host cells and synthesize human cytokines. Second, using gene knock-in methods, immunodeficient mouse strains have been generated to express human major histocompatibility complex (MHC) while suppressing mouse MHC molecules that improved human immune system development [2, 15]. Transgenic expression of human cytokines and growth factors has been also demonstrated to modulate human HSPCs responses . For instance, human stem cell factor, granulocyte-macrophage colony stimulating factor and interleukin-3 genes inserted NOD-scid IL2rγnull (NSG) mice models demonstrated enhanced hematopoietic reconstitution of human HSPCs . Although this approach is promising, it is labor/time-intensive, requires specialized facilities and equipment, and is limited in throughput of genes expressed in a single mouse. Moreover, ubiquitous expression of human molecules could be problematic to interpret experimental outcomes. Lastly, human cells and tissues can be directly introduced into the murine host. For example, human bone marrow stromal cells (hBMSCs) were introduced into a mouse bone marrow cavity by intra-femoral injection. The presence of human stromal cells in a mouse bone marrow increased engraftment of subsequently injected human HSPCs . However, this invasive procedure is compatible with only a few bones which limits the throughput of this approach in supporting human HSPCs function. Human fetal thymic and fetal liver tissues, known sites of early hematopoiesis, have been implanted in an immunodeficient mouse prior to human HSPCs injection. A mouse carrying primary human fetal tissue organoids demonstrated complete hematopoietic reconstitution of human HSPCs . Although this study demonstrated the possibility to recreate a full spectrum human hematopoietic cells in mouse models, human fetal thymus and liver tissues are difficult to widely source making broad adoption unattainable.
In this report, we introduce an integrated bioengineering approach that can increase the throughput of model systems research for human HSPCs by using genetically engineered scaffold microenvironments. Engineered stromal cells, stably synthesizing a human cytokine or growth factor, were embedded in three-dimensional (3D) porous hydrogel scaffolds and implanted into immunodeficient mice in different subcutaneous locations. Each implant created a unique hematopoietic-supportive tissue microenvironment over time. These implants exhibited a locally concentrated environment of human cells and soluble factors with the opportunity to make multiple, autonomous scaffolds implanted into the same mouse. We demonstrated the engineering of stromal cells with individual human cytokines, the control of local and systemic exposure of engineered cytokines, and functional characterization of the implanted microenvironments with human HSPCs. The presented method is versatile and can be readily applied to other human cells and cytokines with minor modifications for better understanding human biology.
All chemicals and supplies were purchased from Sigma Aldrich or Fisher Scientific unless otherwise stated. All mouse and primary human cell experiments were reviewed and approved by an internal review board of Massachusetts General Hospital.
Stromal cell culture
Primary hBMSCs were isolated from healthy donor’s fresh bone marrow (Lonza) following a previously reported protocol . Briefly, hBMSCs were cultured with medium composed of 15 % fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 20 mg/L gentamicin, 1 ng/L fibroblast growth factor, and 3 g/L sodium bicarbonate in alpha-minimum essential medium (a-MEM). Human umbilical vein endothelial cells (HUVECs) were purchased from Invitrogen and cultured with medium composed of 10 % low serum growth supplement, 100 U/mL penicillin and 100 μg/mL streptomycin. Mouse bone marrow stromal cells (mBMSCs) were purchased from Invitrogen and cultured with medium composed of 15 % fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 20 mg/L gentamicin, 1 ng/L fibroblast growth factor, and 3 g/L sodium bicarbonate in a-MEM. All cell cultures were maintained at 37 °C, 5 % CO2 and 100 % humidity. Human peripheral blood derived CD34 cells were purchased from StemCell Technologies and after thawing immediately used for in vivo experiments without culture.
Generation of lentiviral particles encoded human VEGFa, SDF1a and TNFa genes
The self-inactivating lentiviral vector backbone pRRL.PPT.SFFV.IRES.eGFP were kindly provided by Christopher Baum (Hannover Medical School, Hannover, Germany). cDNA clones of the human cytokines Vascular Endothelial Growth Factor A (hVEGFa), Stromal Derived Factor 1 Alpha (hSDF1a), and Tumor Necrosis Factor Alpha (hTNFa) were purchased from Open Biosystems, Polymerize chain reaction (PCR) amplified, and cloned into pRRL.PPT.SFFV.IRES.eGFP. Lentiviral particles were generated by transient calcium phosphate transfection of HEK293T cells (ATCC CRL-3216) with the lentiviral constructs, psPAX2 (Addgene Plasmid #12260), and pMD2.g (VSV-G, Addgene Plasmid #12259) at the ratio 1: 2: 0.33. Viral supernatants were harvested 40 h post transfection by centrifugation and stored −80 °C until use.
Generation of genetically engineered mouse bone marrow stromal cells
mBMSCs were plated in a 12-well plate. Once the culture reached about 50 % confluence, the medium was replaced with 0.5 ml of new medium containing 1:10 diluted lentiviral particles and 2 μg/ml Polybrene (Hexadimethrine bromide), and incubated for 8 h. Afterwards the cell surface was washed with PBS three times and 1 ml of normal medium was added in each well. After 48 h, mBMSCs expressing green fluorescent protein (GFP) were sorted out using a fluorescence activated cell sorting (FACS) machine (BD FACSAria II cell sorter). Expression of GFP was confirmed under a fluorescent microscope (Zeiss Axio 200) and secretion of hVEGFa, hSDF1a, and hTNFa was determined by enzyme-linked immunosorbent assay (ELISA) kits (R&D systems).
Fabrication of inverted colloidal crystal hydrogel scaffolds and in vitro 3D stromal cell culture
Polyacrylamide hydrogel based inverted colloidal crystal scaffolds were prepared following the previously reported methods . Final hydrogel scaffolds were consisted of regularly arranged spherical pores (D = 250 ± 30 μm) of which surface was coated with type I collagen utilizing amine reactive heterbifunctional cross-linker (Sulfo-SANPH). Dimensions of cylindrical shape hydrogel scaffolds were about 1.5 mm in thickness and 6.5 mm in diameter. Prior to cell seeding, hydrogel scaffolds were dehydrated in a laminar hood for 30 min and then a dense genetically engineered mBMSCs or hBMSCs (0.5 × 106 cells in 40 μl) was dropped on the hydrogel scaffold. Rehydration process promoted effective cell distribution into a complex 3D porous geometry and seeded stromal cells formed stable adhesion on collagen fiber coated pore surface in 4–6 h. After 3 days culture, the level of hVEGFa, hSDF1a, and hTNFa molecules in the medium was determined using ELISA kits. Total cell mass in each scaffold was quantified using a MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent (ATCC) and used for normalization of cytokine secretion.
In vivo microenvironments formation with genetically engineered stromal cells
Three stromal cell-seeding compositions for creating 4 distinct microenvironments in a mouse
Implant site 1
Growth-competent mBMSCs secreting hVEGFa
Growth-competent mBMSCs secreting hVEGFa + hBMSCs
Growth-arrested mBMSCs secreting hVEGFa
Implant site 2
Growth-competent mBMSCs secreting hSDF1a
Growth-competent mBMSCs secreting hSDF1a + hBMSCs
Growth-arrested mBMSCs secreting hSDF1a
Implant site 3
Growth-competent mBMSC ssecreting hTNFa
Growth-competent mBMSCs secreting hTNFa + hBMSCs
Growth-arrested mBMSCs secreting hTNFa
Implant site 4
Growth-competent mBMSCs control
Growth-competent mBMSCs control + hBMSCs
Growth-arrested mBMSC control
Systemic migration of human CD34 cells to the implanted microenvironments
A 2:1 ratio mixture of growth-arrested genetically engineered mBMSCs (0.5 × 106 in 40 μl) and hBMSCs (0.25 × 106 in 20 μl) was introduced into a scaffold. Post 3 days in vitro culture, the cell-scaffolds were subcutaneously implanted into NSG mouse. After 4 weeks implantation, 2.5 × 105 human CD34 (hCD34) cells were intravenously injected. Post 24 and 72 h, mice were sacrificed and implanted scaffolds, bone marrow and spleen samples were collected. Hematopoietic cells were retrieved from scaffold and tissue samples following the previously reported method . Briefly, explanted scaffolds were minced into small pieces (1–2 mm), incubated in 2 mg/ml collagenase solution for 20 min and thoroughly mashed using a plunger rod from a 3 ml syringe. 40 μm cell-strainers were used to separate cells from hydrogel scaffold and tissue debris. For bone marrow cell isolation, tibias and femurs were dissected and the marrow space was flushed with medium using a 23-gauge needle. Collected marrow cells were passed through 70 μm cell-strainers to remove bony spicules and debris and treated with red blood cell (RBC) lysis buffer for 1–2 min. For spleen cell isolation, a retrieved spleen was placed on a 40 μm cell-strainer and gently mashed using a plunger rod with Rosewell Park Memorial Institute (RPMI) medium. Harvested spleen cells were treated with RBC lysis buffer for 2–3 min. Retrieved hematopoietic cells were stained with fluorescein isothiocyanate (FITC) conjugated anti-human CD34 antibody (BD Pharmingen) and analyzed using FACS machine (BD) and FlowJo program.
Direct implantation of human CD34 cells with genetically engineered stromal cell-scaffolds and ex vivo methylcellulose assay
A 2:1 ratio mixture of growth-arrested genetically engineered mBMSCs (0.5 × 106 in 40 μl) and hBMSCs (0.25 × 106 in 20 μl) was introduced into a scaffold. Post 3 days culture, cell-scaffold plates were placed in a refrigerator for 20 min and then removed medium both from a well and a hydrogel scaffold using a pipette. Next ice-cold, moderately dehydrated cell-scaffolds were transferred to a 96-well plate and 50 μl of ice-cold matrigel containing the mixture of 2 × 105 HUVECs and 2 × 105 hCD34 cells was infiltrated into the hydrogel scaffold pores via centrifugation at 4 °C, 1500 rpm for 15 min. The matrigel-cell filled scaffolds were incubated for 20 min at 37 °C incubator to solidify the gel and then 1 ml of culture medium was placed in each well.
After 3 weeks in vivo implantation, hematopoietic cells were retrieved from explanted scaffolds following the above procedure. Hematopoietic cells were dispersed in methylcellulose medium containing recombinant cytokines and erythropoieting for human cells (MethoCult H4034, Stem Cell Technologies). Final concentration was 1 × 105 hematopoietic cells in 1.1 ml methylcellulose medium that was plated in a 35 mm dish following a protocol provided by the vendor. After 2 weeks incubation, colony forming units of (i) erythroid, (ii) granulocyte and macrophage, (iii) granulocyte, erythrocyte, monocyte and megakaryocyte, and (iv) burst forming unit of erythroid were distinguished and counted under an inverted light microscope.
Frozen scaffold blocks were cut into 10–30 μm thickness using a cryostat (Leica Biosystems) and stored at −80 °C until use. For hematoxylin and eosin, and Masson’s Trichrome staining, frozen tissue sections were fixed with 10 % buffered formalin solution and stained following the vendor’s protocol (American Master Tech). For immunofluorescence staining, frozen tissue sections were fixed with ice-cold acetone, blocked with 10 % normal goat serum and 1 % bovine serum albumin diluted in phosphate buffered saline (PBS). Slides were incubated with rat anti-mouse CD31 (BD Pharmingen) and rabbit anti-human CD34 antibody (Abcam) for overnight. Subsequently the slides were stained by goat anti-rat immunoglobulin G (IgG) conjugated with alexa fluor 488 and goat anti-rabbit IgG conjugated with alexa fluor 568 (Invitrogen) for 1 h. Finally, VectaShield mounting medium with DAPI was applied and slides were imaged under a fluorescence (Zeiss 200) microscope. Open source image analysis software, ImageJ, was used to process and quantify areas of collagen fibers and vasculatures in Masson’s Trichrome and mouse CD31 stained slides, respectively.
Explanted scaffolds were fixed in 2 % glutaladehyde solution for 6 h and then serially dehydrated with 20, 50, 70, 90 and 100 % ethanol solution. The scaffolds were further dried using a lyophilzer overnight. A thin platinum/gold coating was made on the samples using a sputter coating machine (208HR,Cressington) and imaged under FESEM Ultra55 (Zeiss).
Statistical comparisons of data were per- formed using SPSS version 17 software. Nonparametric tests, i.e., Kruskal-Wallis and Mann-Whitney tests, were applied for ELISA and LSK cell analysis, respectively. Comparisons of human cell engraftment in long-term engraftment studies were performed using an unpaired Student t test on GraphPad PRISM version 5.
Genetically engineered mouse stromal cell lines secreting human VEGFa, SDF1a, or TNFa
Genetically engineered stromal cells were then seeded into the 3D hydrogel scaffolds following the previously reported methods . These hydrogel scaffolds consisted of regularly arranged spherical cavities, whereby the cavity surfaces were coated with type I collagen. This coating method promoted homogenous stromal cell seeding and subsequent adhesion (Fig. 1b). The characterized rate of soluble factor secretion of genetically engineered stromal cells in the scaffolds was 4.42 ± 0.24 μg/mL for hVEGFa, 0.87 ± 0.16 μg/mL for hSDF1a, and 2.7 ± 0.02 μg/mL for hTNFa over 3 days. When compared to primary hBMSCs growing in the scaffolds, normalized hVEGFa and hSDF1a secretion were about 4.8 and 3.7 folds higher, respectively (Fig. 1c-e). hBMSCs do not naturally secrete hTNFa. These stable cell lines were advanced for further in vivo testing.
Control of systemic and local exposure of engineered factors after in vivo implantation
Local tissue formation in scaffolds is altered by stromal cell proliferation
Implanted microenvironments support systemic migration and retention of human CD34 cells
Skewed in vivo differentiation of hCD34 cells in engineered microenvironments
The goal of humanized mouse models is to reconstitute specific cellular and tissue dynamics to study human cell biology in vivo and potentially become a testbed to predict human responses for precision medicine. Studies using immunodeficient mice that accept human cells/tissue have generally shown that by adding complimentary human tissues, a more humanized response can be seen . In this study, we wanted to create a testing platform where molecules of interest to the human hematopoietic system can be studied in a locally enriched environment with easy access and analytical capability. Our hypothesis was that a single mouse can harbor multiple independent microenvironments that have site-specific bioactivity by implanting genetically engineered stromal cell-laden scaffolds. Mouse cells are generally more difficult to infect with lentivirus than human cells, though they are more proliferative after infection and also could survive longer after in vivo implantation than human cells . Our infection efficiency of mouse stromal cells was ~10–30 %. The transfected cells maintained proliferative capacity after purification by flow sorting and expressed properly folded hVEGFa, hSDF1a, and hTNFa. The constitutive translation of these proteins by engineered cells was 4–5 fold more in concentration than normal hBMSCs with respect to hVEGFa and hSDF1a. hTNFa is not naturally expressed by hBMSCs and this construct was included to mirror an inflammatory reaction site. This mouse cell expression was robust and took only a few weeks as opposed to months/years for transgenic mice, which can further benefit from improvement in infection efficiency while exploring new promoter systems and unknown molecules of interest to microenvironment research.
When compared to in vivo gene delivery methods that non-specifically infect host cells to synthesize desired human cytokines, an engineered ectopic tissue analogue is an alternate approach with multiple advantages. First, the porous hydrogel scaffolds were a very useful tool for standardizing and analyzing engineered tissue microenvironments. The fully interconnected microscale cavities made with a synthetic hydrogel matrix promoted angiogenesis and, in turn, improved the survival and function of genetically engineered stromal cells after implantation. These features allowed for direct comparison of the effects of the overexpressed gene on local tissue development and human HSPC fate. Second, these engineered tissue analogues could be studied for local or systemic effects by simply controlling the growth potential of the engineered stromal cells. This also enables an increase in the throughput of studies by implanting several unique microenvironments into a single host mouse. Finally, implanted microenvironments can accommodate human HSPCs and be directly accessed for various imaging and analytical tools [20, 30]. Thus, implantable microenvironments that harbor genetically engineered cells can broaden the study of human cells in immunodeficient mouse models.
In our in vivo studies with engineered stromal cells, we observed systemic levels of expressed cytokine and a mild sarcoma-like overgrowth of these stromal cell lines that were not under growth arrest. A typical fibroblastic compartment is quiescent, unless the tissue bed is activated at which time the cells have been observed to proliferate. The tissue cavity was not considered conducive to support primary functions of hematopoiesis. Instead, a growth-arrested stromal cell population could still express soluble, bioactive factors in an enriched environment while allowing for additional tissue elements (e.g. hBMSCs, HUVECs, hCD34 cells) to be included and maintain viability within the space as seen in Fig. 5. Using growth-arrested stromal cells eliminated systemic levels of expressed cytokines as well. This stromal population was put on test in a competitive transplantation assay and we did not observe site-specific homing of hCD34 cells, even to a potent chemokine hSDF1a. The lack of a systemic chemokine gradient may be a plausible explanation for non-specific homing results and can be an area of improvement with a temporal and stronger burst of cytokine from a non-proliferative cell mass.
The human cytokines overexpressed in our model system, namely hVEGFa, hSDF1a, and hTNFa, were selected based on known biological responses to hematopoietic activity to benchmark our bioactivity studies. hVEGFa is a potent stimulator of angiogenesis and has direct effect on promoting the survival of HSPCs [31, 32]. hSDF1a is a well-known chemokine molecule that actively recruits circulating HSPCs [27, 33, 34] and maintains their activities . hTNFa restricts HSPCs activity functioning as a key precursor for inflammatory response [36, 37]. Although these cytokines are designed for human specific, evolutionary conservation of key structures and functions in these molecules exhibited cross-reactivity with murine tissue/cells that in turn promote distinct tissue microenvironment formation depending on types of engineered stromal cells. We observed sustained vascular area in hVEGFa constructs, more cellularity and progenitor cell activity in hSDF1a constructs, and more inflammatory cell recruitment and collagen remodeling in hTNFa constructs. Our model system further reflected key biological responses in vivo interacting with hCD34 HSPCs. This model system can be deemed suitable to understand short-term effects of engineered human proteins on HSPCs function for future discovery and validation studies. There may also be cross-reactivity of mouse molecules on human HSPCs and vice versa, though we maintained the same cellular composition of our scaffolds to control for this potential effect. A long-term evaluation of these microenvironments is necessary to establish true stem cell functions of serial transplantation, reconstitution, and tri-lineage differentiation after months in vivo.
In conclusion, we presented a bioengineering strategy to improve humanized mouse models by creating implantable microenvironments that release human specific cytokines and growth factors in a controlled manner. Genetically engineered stromal cells stably synthesize encoded human molecules after in vivo implantation. Porous hydrogel scaffolds facilitated the usage of engineered stromal cells as forming tissue-engineered depots that continuously release engineered human molecules. Presented approach is simple and versatile that can be easily applied to other soluble and insoluble molecules. Combination of engineered stromal cells and implantable microenvironments is expected to be a valuable tool for generating humanized mouse models and utilizing them for basic and translational human HSPCs as well as cancer research.
hVEGFa, vascular endothelial growth factor a; hSDF1a, stromal derived factor 1 alpha; hTNFa, tumor necrosis factor alpha; HSPCs, hematopoietic stem and progenitor cells; hBMSCs, human bone marrow stromal cells; mBMSCs, mouse bone marrow stromal cells; HUVECs, human umbilical vein endothelial cells; a-MEM, alpha-minimum essential medium; FACS, fluorescence activated cell sorting; SEM, scanning electron microscopy; ECM, extracellular matrix; CFU, colony forming unit; NSG, NOD-scid IL2rγnull; eGFP, enhanced green fluorescent protein; MHC, major histocompatibility complex; MTT, MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
We give thanks to Drs. Rebekka Schneider-Kramann and Benjamin Ebert for providing reagents for the study and helpful discussions. We also acknowledge the efforts of Jessica Elman and Ryan Murray for assisting lentiviral gene transduction and FACS cell sorting experiments, and tail vein injection of hCD34 cells, respectively.
This work was supported by NIH Grants R01EB012521 (B.P.), K01DK087770 (B.P.), R00CA163671 (J.L.) and the Shriners Hospitals for Children (B.P., J.L.).
Availability of data and materials
Additional file 1: Figure S1-S6 are available on the web.
JL and BP conceived the idea, designed experiments, analyzed data and wrote the manuscript. DH prepared Lentivirus particles. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Authors consent for publication.
Ethics approval and consent to participate
All mouse and primary human cell experiments were reviewed and approved by an internal review board of Massachusetts General Hospital.
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