Efficient Generation of Skin Organoids from Pluripotent Cells via Defined Extracellular Matrix Cues and Morphogen Gradients in a Spindle‐Shaped Microfluidic Device

Skin organoids with inside-out anatomy have to be precisely incised and/or manually inverted to generate functional grafts and models.

“Moreover, skin organoids with inside-out anatomy have to be precisely incised and/or manually inverted to generate functional grafts and models [3,5].”

• cites [3] Lee J, van der Valk WH, Serdy SA, Deakin CC, Kim J, Le AP, Koehler KR, Nat. Protoc. 2022, 17, 1266. 35322210 PMC10461778 sourceDisputed

  The Introduction’s “Limitations” section confirms the inside-out cystic anatomy. But “Skin reconstruction and wound healing” says intact organoids were placed into incisions in mouse skin, then unfurled and integrated after transplantation. It does not describe incising or manually inverting the organoids as necessary to make grafts. (LLM, conf=0.88)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured
• cites [5] Jung S-Y, You HJ, Kim M-J, Ko G, Lee S, Kang K-S, iScience 2022, 25, 105150. 36193049 PMC9526179 sourcePartly supported

  The Introduction describes enclosed skin organoids with inside-out anatomy. In Results > Air-liquid interface (ALI)-skin organoids (page unavailable), the authors report dissecting organoids into four portions and placing them dermis-down to expose the epidermis, producing a skin model. The paper does not establish that precise incision or manual inversion is necessary for functional grafts, and it does not describe generating grafts. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Collagen type I is the major constituent of the dermis, interlaced with other important ECM components such as collagen III and VII, fibronectin, vitronectin, hyaluronic acid, and glycosaminoglycans, such as heparin.

“Unlike Matrigel, the major constituent of the dermis is collagen type I, which is interlaced with other important ECM components such as collagen III and VII [23,24], fibronectin [25], vitronectin [11], hyaluronic acid [26,27], and glycosaminoglycans, such as heparin [28], all of which contribute to key physiological skin functions.”

• cites [23] Smith LT, Holbrook KA, Madri JA, Am. J. Anat. 1986, 175, 507. 3521252 sourceNot checked — no full text of the source
• cites [24] Mouw JK, Ou G, Weaver VM, Nat. Rev. Mol. Cell Biol. 2014, 15, 771. 25370693 PMC4682873 sourceCould not verify

  The citation to this source is attached specifically to vitronectin. In our copy, searches for vitronectin and dermis found vitronectin only in the References section; the substantive ECM passages do not establish that it is a component of the dermis. (LLM, conf=0.72)

  Experimental — accuracy not yet measured
• cites [25] Tracy LE, Minasian RA, Caterson EJ, Adv. Wound Care 2016, 5, 119. PMC4779293 26989578 sourceSupported
• cites [11] Huang J, Heng S, Zhang W, Liu Y, Xia T, Ji C, juan Zhang L, Semin. Cell Dev. Biol. 2022, 128, 137. 35339360 sourceNot checked — no full text of the source

Current PSO protocols generate skin as free-floating cellular aggregates in medium suspension in the absence of physiological 3D ECM cues normally found in skin development.

“Despite the ECM’s critical role during organogenesis, the current PSO protocols generate skin as free-floating cellular aggregates in medium suspension in the absence of physiological 3D ECM cues normally found in skin development.”

• cites [2] Lee J, Bӧscke R, Tang PC, Hartman BH, Heller S, Koehler KR, Cell Rep. 2018, 22, 242. 29298425 PMC5806130 sourcePartly supported

  The Discussion describes floating aggregates, and Experimental Procedures (PSC differentiation; page unavailable) places individual aggregates in low-adhesion wells containing medium. But the paper does not establish that they lack physiological 3D ECM cues: the protocol adds Matrigel, and the Results (Self-assembly of skin organoids recapitulates embryonic development; page unavailable) report ECM proteins at the epidermal–dermal interface. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [3] Lee J, van der Valk WH, Serdy SA, Deakin CC, Kim J, Le AP, Koehler KR, Nat. Protoc. 2022, 17, 1266. 35322210 PMC10461778 sourcePartly supported

  The Materials > Equipment setup procedure describes a transition to floating culture in medium, supporting the free-floating aggregate portion of the claim. But Materials > Reagents setup specifies Matrigel in both the initial differentiation medium (2%) and the floating-culture medium (1%). The paper does not establish that these cultures lack physiological 3D ECM cues, so that part is not supported by this source. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

In mouse PSOs, Atoh1 is expressed in pluripotent cells.

“According to previous studies in mouse PSOs [2], Atoh1 is expressed in pluripotent cells; it switches off with the loss of pluripotency, and then turns back on after day 10 of differentiation as an indicator of epidermal differentiation.”

• cites [2] Lee J, Bӧscke R, Tang PC, Hartman BH, Heller S, Koehler KR, Cell Rep. 2018, 22, 242. 29298425 PMC5806130 sourceCould not verify

  The Results section identifies an Atoh1/nGFP mouse embryonic stem-cell line used to produce skin organoids, but it does not say that Atoh1 is expressed in pluripotent cells. Searches for Atoh1 expression in pluripotent cells and for the claimed differentiation timeline found no passage establishing that expression pattern. (LLM, conf=0.74)

  Experimental — accuracy not yet measured

Formation of necrotic cores was previously observed in other large organoid systems.

“The formation of necrotic cores was previously observed in other large organoid systems [19].”

• cites [19] Kozlowski MT, Crook CJ, Ku HT, Commun. Biol. 2021, 4, DOI 10.1038/s42003-021-02910-8. 34893703 PMC8664924 sourcePartly supported

  In “Synthetic hydrogels in action: Modeling difficult tissues such as the brain and pancreas” (page unavailable), the review says larger islets are known to form necrotic centers because oxygen and nutrients cannot diffuse sufficiently. It raises this as a concern while discussing the size of islet-organoid aggregates, but does not say necrotic cores were observed in those organoids or in other large organoid systems. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

Many other organoid models, including brain, intestinal, mammary gland, and retinal organoids, employ 3D ECM-based hydrogels to induce complex tissue architecture that cannot be achieved in ECM-free floating culture.

“While many other organoid models, including brain[13], intestinal [14], mammary gland [15], and retinal organoids [16], employ 3D ECM-based hydrogels to induce complex tissue architecture that cannot be achieved in ECM-free floating culture, the same has not been explored for skin yet.”

• cites [13] Cho AN, Jin Y, An Y, Kim J, Choi YS, Lee JS, Kim J, Choi WY, Koo DJ, Yu W,, Nat. Commun. 2021, 12, DOI 10.1038/s41467-021-24775-5. 34354063 PMC8342542 sourcePartly supported

  The Results describe embedding brain organoids in a 3D ECM hydrogel, and the Discussion reports more complex brain-like structures with the ECM–microfluidic platform. The paper does not establish that those structures cannot be achieved in ECM-free floating culture; the reported platform also combines the hydrogel with microfluidics. Citation [13] is attached to the brain-organoid example, not the other organoid types. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [14] Rezakhani S, Gjorevski N, Lutolf MP, Biomaterials 2021, 276, DOI 10.1016/j.biomaterials.2021.121020. 34280822 sourcePartly supported

  Citation [14] is attached to intestinal organoids. Section 3.2 (page unavailable) says intestinal organoid cultures rely on Matrigel and describes hydrogels that support intestinal morphogenesis. It does not establish the claimed comparison that complex architecture cannot be achieved in ECM-free floating culture; the other organoid types have separate citations. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [15] Buchmann B, Engelbrecht LK, Fernandez P, Hutterer FP, Raich MK, Scheel CH, Bausch AR, Nat. Commun. 2021, 12, DOI 10.1038/s41467-021-22988-2. 33980857 PMC8115695 sourcePartly supported

  The Results section reports that human mammary organoids grown in freely floating collagen I gels develop branches and mammary-like bilayered architecture. This supports the mammary-organoid example, but the paper does not establish that such architecture cannot be achieved in ECM-free floating culture. The other organoid types have separate citations in the citing sentence. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Working collagen I concentrations at physiological levels do not recapitulate the mechanical properties of the native ECM.

“However, working collagen I concentrations at physiological levels[59,62,63] do not recapitulate the mechanical properties of the native ECM[64–66].”

• cites [59] Antoine EE, Vlachos PP, Rylander MN, Tissue Eng. - Part B Rev. 2014, 20, 683. PMC4241868 24923709 sourceSupported
• cites [62] Dinescu S, Albu Kaya M, Chitoiu L, Ignat S, Kaya DA, Costache M, in Cellul. Superabsorbent Hydrogels (Ed: Mondal MIH), Springer International Publishing, Cham, 2019, pp. 1643– 1664.Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [63] Sander EA, Lynch KA, Boyce ST, J. Biomech. Eng. 2014, 136, DOI 10.1115/1.4026290. PMC4023834 24356985 sourcePartly supported

  The Introduction reports that many skin substitutes do not recover the mechanical properties of native skin, and the Mechanical Properties section measures the stiffness of a collagen-based scaffold and engineered skin constructs. But this study uses a collagen–chondroitin-sulfate scaffold; it does not establish that working collagen I concentrations at physiological levels fail to reproduce native ECM mechanics. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [64] Griffin MF, Leung BC, Premakumar Y, Szarko M, Butler PE, Otolaryngol J. - Head Neck Surg. 2017, 46, DOI 10.1186/s40463-017-0210-6. PMC5395887 28420435 sourcePartly supported

  The Results (Biomechanical testing; page unavailable) report Young’s elastic moduli for native skin at several sites, providing measurements of native-tissue mechanical properties. The paper does not test working collagen I concentrations or compare collagen I materials with native ECM, so it does not establish that they fail to recapitulate those properties. (LLM, conf=0.79)

  Experimental — accuracy not yet measured
• cites [65] Pailler-Mattei C, Bec S, Zahouani H, Med. Eng. Phys. 2008, 30, 599. 17869160 sourceNot checked — no full text of the source
• cites [66] Delalleau A, Josse G, Lagarde JM, Zahouani H, Bergheau JM, Biomech J. 2006, 39, 1603. 15990103 sourceNot checked — no full text of the source

The presence of COL I+LAM511 hydrogel is a more efficient system to induce hair follicle formation in PSOs compared to previous methods.

“Our data showed that the presence of COL I+LAM511 hydrogel is a more efficient system to induce hair follicle formation in PSOs, compared to the previous methods [2,3], however, the variability in hair forming efficiency draws attention to the need to further engineer the PSO microenvironment.”

• cites [2] Lee J, Bӧscke R, Tang PC, Hartman BH, Heller S, Koehler KR, Cell Rep. 2018, 22, 242. 29298425 PMC5806130 sourceSupported
• cites [3] Lee J, van der Valk WH, Serdy SA, Deakin CC, Kim J, Le AP, Koehler KR, Nat. Protoc. 2022, 17, 1266. 35322210 PMC10461778 sourceSupported

The spindle-shaped hydrogel culture system led to a two-fold increase in keratinocyte differentiation.

“This new culture system significantly reduced organoid necrosis, and led to a two-fold increase in keratinocyte differentiation and an eight-fold increase in hair follicle formation.”

The microfluidic system improved spatial organization of dermal and epidermal cells.

“Finally, we adapted the system as a microfluidic device to create asymmetrical gradients of differentiation factors, and improved spatial organization of dermal and epidermal cells.”

The microfluidic device creates asymmetrical gradients of differentiation factors.

“Finally, we adapted the system as a microfluidic device to create asymmetrical gradients of differentiation factors, and improved spatial organization of dermal and epidermal cells.”

PSOs generated in collagen type I supplemented with laminin 511 exhibit increased growth compared to conventional free-floating condition.

“After examining various dermal ECM molecules, we found that PSOs generated in collagen type I supplemented with laminin 511 exhibit increased growth compared to conventional free-floating condition, but fail to induce complete skin differentiation due in part to necrosis.”

The 3D bioprinted spindle-shaped hydrogel device constrains organoid growth longitudinally.

“We addressed this problem by generating the PSOs in a 3D bioprinted spindle-shaped hydrogel device, which constrains organoid growth longitudinally.”

The spindle-shaped hydrogel culture system led to an eight-fold increase in hair follicle formation.

“This new culture system significantly reduced organoid necrosis, and led to a two-fold increase in keratinocyte differentiation and an eight-fold increase in hair follicle formation.”

The spindle-shaped hydrogel culture system significantly reduced organoid necrosis.

“This new culture system significantly reduced organoid necrosis, and led to a two-fold increase in keratinocyte differentiation and an eight-fold increase in hair follicle formation.”

There is a significant difference in GFP expression per area for COL I+LAM511 hydrogels (28%) compared to Matrigel (13%) or COL I alone (23%).

“Nonetheless, there is a significant difference for COL I+LAM511 hydrogels (28%) when compared to those of Matrigel (13%) or with only COL I (23%), which may indicate higher efficiency of differentiation towards epidermal progenitors.”

Overall GFP expression per area generally increased for EBs encapsulated within COL I matrices, with or without other molecules, compared to floating conditions or Matrigel.

“In terms of overall GFP expression, our data shows a general increase in the GFP expression per area for EBs encapsulated within COL I matrices (in the presence or the absence of other molecules) when compared to the floating conditions or in the presence of Matrigel (Figure 2E).”

In the authors' experiments, PSOs displayed different patterns and levels of differentiation in response to ECM type.

“Interestingly, in our experiments, PSOs displayed different patterns and levels of differentiation in response to ECM type (Figure 2B).”

PSO growth was enhanced in an ECM-specific manner.

“Quantification of the cross-sectional area on day 14 showed that PSO growth was enhanced in an ECM-specific manner (Figure 3A).”

Matrigel alone produced high variability in the level and pattern of GFP expression.

“Notably, Matrigel alone produced high variability in the level and pattern of GFP expression, consistent with its undefined nature and inherent variability.”

In numerical simulations using COMSOL, the majority of ECM-embedded spherical PSOs >1 mm in diameter were hypoxic (<5%) with core oxygen <0.2% O2, within necrotic range.

“Using oxygen (O2) as the model molecule, we found that the majority of the PSOs were hypoxic (<5%) where the core was deprived of oxygen (<0.2% O2; within necrotic range) in spherical PSOs >1 mm in diameter.”

The number of hair follicles present in each PSO is considerably higher for PSO culture in the spindle device.

“Moreover, the number of hair follicles present in each PSO is considerably higher for PSO culture in the spindle device (Figure 5G), highlighting that this culture approach combined with the presence of COL I+LAM511 hydrogel boosts hair follicle differentiation.”

Spindle culture in COL I+LAM511 increased adipocyte differentiation compared to spherical COL I+LAM511 culture.

“In addition, spindle culture in COL I+LAM511 increased adipocyte differentiation compared to spherical COL I+LAM511 culture (Figure 5H–I).”

Relative gene expression of hair follicle markers Krt71 and AE13 was improved.

“This was further supported by improved relative gene expression of hair follicle markers, Krt71 and AE13 (Figure S4B).”

The microfluidic device can generate steep chemical gradients of both high and low molecular weight molecules across the middle culture channel.

“Fluorescent intensity signal across the PSO culture chamber at different timepoints (Figure S5) shows the capability of the device to generate steep chemical gradients of both high (Figure 6A) and low (Figure 6B) molecular weight molecules across the middle culture channel.”

In spindle-shaped PSOs, O2 concentration ranged from 10 to 1.5% O2, not reaching critically low levels when PSO width was constrained below 500 μm, regardless of length.

“In contrast, O2 concentration in spindle-shaped PSOs ranged from 10 to 1.5 % O2, not reaching critically low levels of O2 when the PSO width was constrained below 500 μm, regardless of the length of the PSO.”

When PSOs were encapsulated within Matrigel or COL I+LAM511 in the microfluidic device, they showed lower cell death compared to gel-spheroid culture condition.

“Similarly, when PSOs were encapsulated within Matrigel or COLI+LAM511 in the microfluidic device, they showed lower cell-death compared to gel-spheroid culture condition (Figure 7C), consistent with the previous result for the spindle-device (Figure 4E–G).”

The spindle-culture method enabled hair follicle formation in the presence of melanocytes and pigmentation.

“The spindle-culture method not only enabled hair follicle formation in the presence of melanocytes and pigmentation [45,46] but also significantly promoted the efficiency of hair follicle formation up to 80% efficiency when compared to other culture conditions (Figure 5B–F).”

• cites [45] Müller-Röver S, Tokura Y, Welker P, Furukawa F, Wakita H, Takigawa M, Paus R, Exp. Dermatol. 1999, 8, 237. 10439220 sourceNot checked — no full text of the source
• cites [46] Patel D, Bassi R, Hooper AT, Sun H, Huber J, Hicklin DJ, Kang X, Anticancer Res. 2008, 28, 2679. 19035294Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

EBs in COL I+LAM511 grew in the spindle chamber by elongating and taking on the spindle shape, reaching ~15 times their initial surface area in 24 days.

“We found that EBs in COL I+LAM511 grew in the spindle chamber by elongating and taking on the spindle shape of the chamber (Figure 4B), reaching ~15 times their initial surface area in 24 days (Figure 4C).”

The spindle-culture method significantly promoted the efficiency of hair follicle formation up to 80% efficiency when compared to other culture conditions.

“The spindle-culture method not only enabled hair follicle formation in the presence of melanocytes and pigmentation [45,46] but also significantly promoted the efficiency of hair follicle formation up to 80% efficiency when compared to other culture conditions (Figure 5B–F).”

Live imaging of GFP-Atoh1 expression in PSOs indicated that most cells lost their pluripotency by day 7, evidenced by loss of GFP signal.

“Live imaging of GFP-Atoh1 expression in PSOs indicated that most cells lost their pluripotency by day 7, evidenced by the loss of GFP signal (Figure 7D and Figure S6).”

Analysis of two independent batches with over 10 PSOs per condition consistently shows that spindle shape culture of PSOs promotes hair follicle formation more efficiently compared to conventional culture conditions in both the presence and absence of ECM.

“Analysis of two independent batches of experiments with over 10 PSOs per condition consistently show that the spindle shape culture of PSOs promotes hair follicle formation more efficiently compared to conventional culture conditions in both the presence and absence of ECM.”

In a HUVEC spheroid sprouting assay, cells sprouted from the spheroid towards high VEGF concentration.

“Fluorescence images at different timepoints showed the cells sprouting from the spheroid towards high VEGF concentration (Figure 6D).”

When both BMP4 and LDN concentrations were high, K14+ epidermal cells formed at the center of PSOs, similar to the conventional PSO protocol and spindle device without morphogen gradients.

“When both BMP4 and LDN concentrations were high, the K14+ epidermal cells formed at the center of PSOs, similar to the conventional PSO protocol and our data in spindle device without morphogen gradients (Figure 5).”

PSOs grew in the microfluidic device by elongating and taking the spindle shape of the chamber.

“Allowing the PSOs to mature for 32 days, we found that PSOs grew in the microfluidic device by elongating and taking the spindle shape of the chamber (Figure 7B), similar to the earlier experiments in the spindle device (Figure 4B).”

There was no difference in the total amount of quantified oil-red-o staining between spindle culture in COL I+LAM511 and floating culture.

“Although there was no difference in the total amount of quantified oil-red-o staining compared to floating culture, spindle culture in COL I+LAM511 resulted in generation of adipose tissue as thick layer over the entire PSO (Figure 5H–I), whereas floating culture generated sparse adipose tissue preferentially located near the hair follicles (Figure 3D).”

Spindle COL I+LAM511 culture produced lower numbers of cells in early and late apoptosis compared with floating culture or spherical COL I+LAM511 culture.

“Quantification of the data showed that spindle COL I+LAM511 culture produced lower numbers of cells in early and late apoptosis compared with floating culture or spherical COL I+LAM511 culture (Figure 4E–F).”

PSOs generated in collagen type I supplemented with laminin 511 fail to induce complete skin differentiation due in part to necrosis.

“After examining various dermal ECM molecules, we found that PSOs generated in collagen type I supplemented with laminin 511 exhibit increased growth compared to conventional free-floating condition, but fail to induce complete skin differentiation due in part to necrosis.”

ECM and morphogen gradients play a pivotal role in promoting and spatially-controlling skin differentiation in the PSO framework.

“Our study highlights the pivotal role of ECM and morphogen gradients in promoting and spatially-controlling skin differentiation in the PSO framework.”

The study established a method to generate PSOs effectively in a chemically-defined 3D ECM environment.

“In this work, we established a method to generate PSOs effectively in a chemically-defined 3D ECM environment.”

ECM cues can be leveraged to enhance the efficiency of epidermal and hair follicle differentiation in PSOs.

“Our study proposed a novel culture method using a 3D bioprinted spindle-shaped hydrogel device for the generation of PSOs in a chemically-defined 3D ECM environment, highlighting that ECM cues can be leveraged to enhance the efficiency of epidermal and hair follicle differentiation in PSOs.”

The study proposed a novel culture method using a 3D bioprinted spindle-shaped hydrogel device for the generation of PSOs in a chemically-defined 3D ECM environment.

“Our study proposed a novel culture method using a 3D bioprinted spindle-shaped hydrogel device for the generation of PSOs in a chemically-defined 3D ECM environment, highlighting that ECM cues can be leveraged to enhance the efficiency of epidermal and hair follicle differentiation in PSOs.”

ECM acts as a molecular reservoir of biological factors, e.g., growth factors, to enhance cell signaling.

“In addition to its structural role in tissue architecture, ECM acts as a molecular reservoir of biological factors, e.g., growth factors, to enhance cell signaling [12].”

• cites [12] Nyström A, Bruckner-Tuderman L, Semin. Cell Dev. Biol. 2019, 89, 136. 30076963 sourceNot checked — no full text of the source

Cell-ECM interactions in early PSO development play a major role in the overall and spatial differentiation of embryonic stem cells.

“This evidence suggested that cell–ECM interactions in early PSO development play a major role in the overall and spatial differentiation of embryonic stem cells.”

The lack of direct external access to the epidermis greatly limits the usefulness of skin organoids in research on skin barrier function, topical drug development, and the skin microbiome.

“The resulting lack of direct external access to the epidermis greatly limits the usefulness of skin organoids in research on skin barrier function, topical drug development, and the skin microbiome.”

• AssumptionRests on an assumption: Skin organoids have inverted anatomy, with the epidermis on the interior and the dermis on the exterior.

ECM cues are important to control PSO generation.

“The dependence of early differentiation on ECM type indicates that ECM cues are important to control PSO generation.”

The failure of COL I+LAM511 to promote complete skin differentiation is probably due in part to nutrient deprivation driven by its larger size.

“Ultimately, while COL I+LAM511 provided a favorable environment for EBs growth and stemness, it failed in promoting complete skin differentiation when compared to floating and Matrigel culture conditions, probably due in part to nutrient deprivation driven by its larger size, which we investigated next.”

COL I+LAM511 failed to promote complete skin differentiation when compared to floating and Matrigel culture conditions.

“Ultimately, while COL I+LAM511 provided a favorable environment for EBs growth and stemness, it failed in promoting complete skin differentiation when compared to floating and Matrigel culture conditions, probably due in part to nutrient deprivation driven by its larger size, which we investigated next.”

The significant difference in GFP expression may indicate higher efficiency of differentiation towards epidermal progenitors for COL I+LAM511.

“Nonetheless, there is a significant difference for COL I+LAM511 hydrogels (28%) when compared to those of Matrigel (13%) or with only COL I (23%), which may indicate higher efficiency of differentiation towards epidermal progenitors.”

The spindle-shaped culture system enhanced follicular and interfollicular epidermal differentiation surrounded by a thick layer of adipose tissue.

“This in turn enhanced follicular and interfollicular epidermal differentiation surrounded by a thick layer of adipose tissue.”

Limited nutrient availability causes failure of ECM-embedded PSOs in later stages.

“Measurement of lactate dehydrogenase (LDH) and PrestoBlue activity in the culture medium (Figure S3B–C) indicated that spherical COL I+LAM511 culture led to cell death, and hence an increase in the death ratio (Figure 4G), supporting our hypothesis that limited nutrient availability causes failure of ECM-embedded PSOs in later stages.”

The data showed the ability to spatially control dermal and epidermal differentiation in PSOs through creating morphogen gradients in the microfluidic spindle device.

“This data showed the ability to spatially-control the dermal and epidermal differentiation in PSOs through creating morphogen gradients in our microfluidic spindle device and highlighted the intricate balance between BMP4 and LDN concentrations in the regulation of this process.”

• AssumptionRests on an assumption: BMP4/SB promote epidermal differentiation and FGF-2/LDN promote dermal differentiation.

The study showed successful generation of PSOs in a defined 3D ECM microenvironment.

“In summary, we showed the successful generation of PSOs in a defined 3D ECM microenvironment.”

The data highlighted the intricate balance between BMP4 and LDN concentrations in the regulation of dermal and epidermal differentiation.

“This data showed the ability to spatially-control the dermal and epidermal differentiation in PSOs through creating morphogen gradients in our microfluidic spindle device and highlighted the intricate balance between BMP4 and LDN concentrations in the regulation of this process.”

• AssumptionRests on an assumption: BMP4/SB promote epidermal differentiation and FGF-2/LDN promote dermal differentiation.

The spindle culture approach combined with COL I+LAM511 hydrogel boosts hair follicle differentiation.

“Moreover, the number of hair follicles present in each PSO is considerably higher for PSO culture in the spindle device (Figure 5G), highlighting that this culture approach combined with the presence of COL I+LAM511 hydrogel boosts hair follicle differentiation.”

The spatial pattern of GFP-Atoh1 expression suggests the effect of morphogen gradients at early time points.

“GFP-Atoh1 expression (indicative of epidermal progenitor differentiation [2]) was reestablished after day 14, and remarkably, the expression was located in areas close to the epidermal channel, and was absent near the dermal channel, suggesting the effect of morphogen gradients at early time points.”

Spindle culture can reduce the level of apoptosis in ECM-embedded skin PSOs.

“Our data confirmed that the spindle culture can reduce the level of apoptosis in ECM-embedded skin PSOs.”

Spatial control of dermal and epidermal differentiation in PSOs is possible through creating morphogen gradients using microfluidics.

“In addition, our study suggested that spatial control of dermal and epidermal differentiation in PSOs is possible through creating morphogen gradients using microfluidics.”

• AssumptionRests on an assumption: BMP4/SB promote epidermal differentiation and FGF-2/LDN promote dermal differentiation.